The influence of detergent, the active component of detergent and sodiumtripolyphosphate on the biochemical process of some fungi.

Detergent "Merix" (Merima-Krusevac) and its components, sodiumtripolyphosphate and ethoxyled oleyl-cetyl alcohol in defined concentrations have influence on the enzymatic activity, bioproduction of amino acids and proteins and total biomass of species Aspergillus niger (A. niger) and Fusarium oxysporum (F. oxysporum). Detergent in concentration of 1% vol., in some cases, significantly stimulated production of the majority of amino acids. Detergent and its components (ethoxyled oleyl-cetyl alcohol and sodiumtripolyphosphate) in concentration of 1% vol. showed different influences on the production of proteins by the species of fungi A. niger and F. oxysporum. The enzymatic activity of fungi A. niger and F. oxysporum changed from total inhibition up to partial or total stimulation under influence of investigated pollutants. Detergent, sodiumtripolyphosphate and ethoxyled oleyl-cetyl alcohol, all in concentration of 1% vol. have reduced the production of the biomass of fungi A. niger and F. oxysporum. Investigated fungi have shown different response to the used pollutants.     

Assessment of diversity,distribution and antibacterial activity of endophytic fungi isolated from a medicinal plant <Emphasis Type="Italic">Adenocalymma alliaceum</Emphasis> Miers

A study was conducted for isolation, identification and antibacterial potential of fungal endophytes of Adenocalymma alliaceum Miers., (Bignoniaceae), a medicinal shrub vine plant which has long history for its usages in curing various disorders. A total of 149 isolates of endophytic fungi representing 17 fungal taxa were obtained from 270 segments (90 from each stem, leaf and petiole) of this plant. Hyphomycetes (77.85%) were the most prevalent, followed by Ascomycetes (8.05%) and Coelomycetes (4.03%) respectively. A considerable amount of fungal isolates was kept under (10.07%) Mycelia-Sterilia (MS). Leaf harboured maximum colonization of endophytic fungi (72.22%) which was greater than stem (67.78%) and petiole (25.54%). The Jc similarity index was maximum (0.619) between stem vs leaf followed by leaf vs petiole (0.571) and stem vs petiole (0.428). The dominant endophytic fungi were Alternaria alternata, Aspergillus niger, Stenella agalis, Fusarium oxysporum, Curvularia lunata and Fusarium roseum. Among twelve endophytic fungi tested for antibacterial activity, crude extracts of nine endophytic fungi (75%), showed antibacterial potential against one or more clinical human pathogens. Alternaria alternata, Curvularia lunata, Penicillium sp. and Chaetomium globosum exhibited significant antibacterial activity against 4 of 5 tested pathogens, showing broad spectrum activity. This investigation explains the value of sampling from different tissues of a host plant for the greater species diversity, and additionally, the antibacterial screening of some endophytic fungi from this specific medicinal plant may represent a unique source for many of the useful antibacterial compounds.     

The ability of filamentous fungi to produce acids on indoor building materials

Sixty two filamentous fungi isolated from paint coatings, wallpaper, carton-gypsum board, and indoor air in buildings were screened for acid activity. It was found that 64.5% of strains produce acids on medium with bromo-cresol purple, where 18% of the strains were distinguished by very high acid activity (acid activity coefficient Q = 1.32–2.83), including the species:Aspergillus niger, Aspergillus versicolor, Penicillium expansum, Penicillium brevicom pactum, Penicillium chrysogenum, Cladosporium cladosporioides, Stachybotrys chartarum, Mucor globosus, Ulocladium chartarum andAlternaria alternata. Research indicated that filamentous fungi considerably decrease the pH of the medium when that medium containing building material. The greatest acid production and pH decrease of the medium was observed during the growth of filamentous fungi in a medium with mortar, while the production of acids was less in a medium with cartongypsum board, gypsum, and wallpaper. Filamentous fungi produced succinic, oxalic, malic and fumaric acids in the medium with indoor building materials. It was stated that the type of building material affects the spectrum and quantity of organic acids produced by filamentous fungi.     

Survival of spores of Rhizopus stolonifer, Aspergillus niger, Botrytis cinerea and Alternaria alternata after exposure to ethanol solutions at various temperatures

AIMS: To quantify and model the toxicity of brief exposures of spores of Rhizopus stolonifer, Aspergillus niger, Botrytis cinerea and Alternaria alternata to heated, aqueous ethanol solutions. These fungi are common postharvest decay pathogens of fresh grapes and other produce. Sanitation of produce reduces postharvest losses caused by these and other pathogens. METHODS AND RESULTS: Spores of the fungi were exposed to solutions containing up to 30% (v/v) ethanol at 25-50 degrees C for 30 s, then their survival was determined by germination on semisolid media. Logistical, second-order surface-response models were prepared for each fungus. Subinhibitory ethanol concentrations at ambient temperatures became inhibitory when heated at temperatures much lower than those that cause thermal destruction of the spores by water alone. At 40 degrees C, the estimated ethanol concentrations that inhibited the germination of 50% (LD(50)) of the spores of B. cinerea, A. alternata, A. niger and R. stolonifer were 9.7, 13.5, 19.6 and 20.6%, respectively. CONCLUSIONS: Ethanol and heat combinations were synergistic. Control of spores of these fungi could be accomplished with much lower temperatures and ethanol concentrations when combined compared with either used alone. Botrytis cinerea and A. alternata were less resistant to the combination than A. niger or R. stolonifer.     

Hydrolytic enzymes and antifungal compounds produced by Tilletiopsis species, phyllosphere yeasts that are antagonists of powdery mildew fungi

Isolates of five species of the yeast-like fungus Tilletiopsis Derx (Tilletiopsis albescens Gokhale, Tilletiopsis fulvescens Gokhale, Tilletiopsis minor Nyland, Tilletiopsis pallescens Gokhale, and Tilletiopsis washingtonensis Nyland) were screened for exo- and endo--beta-1,3-glucanase and chitinase production in a liquid broth used to produce inoculum for biological control studies. There were significant differences among the species, and highest overall enzyme activity was present in T albescens and T. pallescens and lowest in T. washingtonensis. A time-course study of beta-1,3-glucanase and chitinase production in T pallescens ATCC 96155 in broth culture with 2.5% glucose as the carbon source showed that enzyme activity gradually increased over a 3- to 21-day period. Maximum enzyme activity was found between pH 4.0 and 5.0. SDS-PAGE of beta-1,3-glucanase isozymes revealed a range of molecular masses from 18 to 29 kDa. Five isozymes were present in both T albescens and T. pallescens and two in T washingtonensis. Antifungal compounds were also detected in ethyl acetate extracts of culture filtrates of T. pallescens ATCC 96155 after 6 days of incubation, while no activity was detected at 14 days. One active fraction was selected following fractionation and preparative chromatography and was bioassayed against Podosphaera (sect. Sphaerotheca) xanthii (Castagne) U. Braun & N. Shishkoff and a number of other fungi. A concentration of 130 microg/mL inhibited germ tube development in P. xanthii, and mildew spores appeared plasmolyzed. Other fungi were inhibited at higher concentrations. Collapse of hyphae and conidiophores was also observed on mildewed leaves treated with the active fraction. Proton NMR analysis indicated that the inhibitory compound was a fatty acid ester. In 3- to 6-day-old cultures of T pallescens ATCC 96155 demonstrating biological control activity, antifungal compound production may have a primary role in restricting growth of mildew fungi and other competitors when applied to leaves.     

Characterization and biocontrol ability of fusion chitinase in Escherichia coli carrying chitinase cDNA from Trichothecium roseum

The antifungal mechanism of mycoparasitic fungi involves fungal cell wall degrading enzymes such as chitinases. Trichothecium roseum is an important mycoparasitic fungus with significant antifungal ability, but studies on chitinases of T. roseum were poor. Here, we report a novel chitinase cDNA isolated from T. roseum by PCR amplification based on conserved chitinase sequences. Southern blot analysis suggested that a single copy of the gene exists in the genome of T. roseum. The deduced open reading frame of 1,143 nucleotides encodes a protein of 380 amino acids with a calculated molecular weight of 41.6 kDa. The fusion chitinase expressed in Escherichia coli has been purified by single-step chromatography. It has a pI of pH 5.4 and expresses a thermal stability, but is insensitive to pH in a broad pH range. According to expectation, E. coli efficiently yielded a high amount of active chitinase. Remarkably, the fusion chitinase offered high antifungal activity.     

里氏木霉与黑曲霉混合发酵产纤维素酶及其水解特性

研究了利用里氏木霉和黑曲霉混合培养产纤维素酶,以黑曲霉孢子悬浮液的不同活化浓度及不同的活化时间来寻找2个菌种发挥最大协同作用的结合点以及所产纤维素酶的水解特性。以里氏木霉单一培养和黑曲霉单一培养为参照进行对比研究。底物为农林废弃物之一的玉米秸秆,经过蒸气爆破预处理后,用作产酶C源。结果表明:黑曲霉孢子悬浮液活化浓度为10个/mL,活化时间为12 h时,滤纸酶比酶活最高,达3.32 U/mL,高于里氏木霉单一培养的2.25 U/mL,β-葡萄糖苷酶比酶活达1.32 U/mL,高于里氏木霉单一培养的0.57 U/mL。为进一步验证混合菌产纤维素酶的水解效果,利用混合菌产纤维酶的酶液及里氏木霉产纤维素酶的酶液进行酶水解实验,当酶用量为20 U/g绝干纤维素,底物质量浓度为100 g/L条件下水解48 h,混合菌所产酶液酶解得率达70.00%,高于里氏木霉所产酶液的酶解得率63.05%。实验表明里氏木霉与黑曲霉混合培养产酶是可行的,并优于单一菌种培养。     

混菌发酵改良棉粕蛋白工艺及协同作用研究

利用筛选到的Aspergillus niger P1和Bacillus subtilis H1混菌发酵改良棉粕蛋白,以提高其利用率。结果表明,混菌发酵的最佳发酵条件为:棉粕 40 g,硫酸铵0.2%,麸皮15%,含水量50%,接种量 15%,同时接菌且两菌接菌比例(A.niger P1 ∶ B.subtilis H1)为2 ∶ 1,发酵温度30 ℃,pH约为6.0,发酵时间 60 h。在此条件下发酵后棉粕小肽含量可提高至18.36%,平均肽链长度可降至4.23,体外消化率可提高至88.59%,显著提高了改良棉粕蛋白的效果。在相同发酵条件下比较单、混菌发酵过程的各营养指标发现:A.niger P1可分泌丰富的酸性蛋白酶,B.subtilis H1可分泌丰富的中、碱性蛋白酶,混菌发酵各种蛋白酶的活性显著提高。混菌发酵将大多数大于10个氨基酸组成的多肽降解为1~3个氨基酸组成的小肽,降解效果明显优于单菌发酵。两菌株具有很好的"协同性",可充分利用棉粕基质,协同改良棉粕蛋白。     

Overproduction and high-yield purification of phospholipase A from the outer membrane of Escherichia coli K-12

The cloned gene for the outer-membrane-bound phospholipase A from Escherichia coli was placed under control of the strong PL promoter of phage lambda. Induction of PL resulted in a 250-fold overexpression up to about 2% total cellular protein. This overproduced enzyme was indistinguishable from the wild-type enzyme. A homogeneous phospholiphase A preparation was obtained in high yield from overproducing bacteria, using the zwitterionic detergent C12-Sulfobetaine and anion-exchange chromatography. Detergent gradients were found to exert great influence on the elution characteristics. Considerations for the choice of optimal detergent gradients are discussed. The purified enzyme migrated as a single 29-kDa band in SDS/polyacrylamide gels, and required Ca(II) for activity. Maximum activity was displayed by enzyme samples taken from solutions with detergent concentrations near the critical micelle concentration. However, upon switching from high to optimal detergent concentration, maximum activity was restored in several hours, probably reflecting a slow conformational transition of the protein. Because the final pure protein was found to hydrolyze phospholipids in the intact erythrocyte membrane, a densely packed bilayer, we assume that this protein is in its biological native state.     

Rational redesign of glucose oxidase for improved catalytic function and stability

Glucose oxidase (GOx) is an enzymatic workhorse used in the food and wine industries to combat microbial contamination, to produce wines with lowered alcohol content, as the recognition element in amperometric glucose sensors, and as an anodic catalyst in biofuel cells. It is naturally produced by several species of fungi, and genetic variants are known to differ considerably in both stability and activity. Two of the more widely studied glucose oxidases come from the species Aspergillus niger (A. niger) and Penicillium amagasakiense (P. amag.), which have both had their respective genes isolated and sequenced. GOx from A. niger is known to be more stable than GOx from P. amag., while GOx from P. amag. has a six-fold superior substrate affinity (K(M)) and nearly four-fold greater catalytic rate (k(cat)). Here we sought to combine genetic elements from these two varieties to produce an enzyme displaying both superior catalytic capacity and stability. A comparison of the genes from the two organisms revealed 17 residues that differ between their active sites and cofactor binding regions. Fifteen of these residues in a parental A. niger GOx were altered to either mirror the corresponding residues in P. amag. GOx, or mutated into all possible amino acids via saturation mutagenesis. Ultimately, four mutants were identified with significantly improved catalytic activity. A single point mutation from threonine to serine at amino acid 132 (mutant T132S, numbering includes leader peptide) led to a three-fold improvement in k(cat) at the expense of a 3% loss of substrate affinity (increase in apparent K(M) for glucose) resulting in a specify constant (k(cat)/K(M)) of 23.8 (mM(-1) · s(-1)) compared to 8.39 for the parental (A. niger) GOx and 170 for the P. amag. GOx. Three other mutant enzymes were also identified that had improvements in overall catalysis: V42Y, and the double mutants T132S/T56V and T132S/V42Y, with specificity constants of 31.5, 32.2, and 31.8 mM(-1) · s(-1), respectively. The thermal stability of these mutants was also measured and showed moderate improvement over the parental strain.     

一株具有拮抗作用的解淀粉芽孢杆菌的筛选、鉴定及生物学特性研究

本研究以樱桃番茄成熟红果为样本,以极细链格孢菌为指示菌筛选得到一株对番茄采后病原菌有抑制作用的菌株KL-1。通过平板拮抗试验研究了菌株KL-1对番茄采后常见病原真菌极细链格孢菌、黑曲霉、青霉菌、尖侧多隔孢霉、拟康宁木霉、盐生枝孢霉、子囊菌、胶孢炭疽菌等八种病原菌的抑制作用,同时通过形态学、生理生化及16S rDNA分子生物学特征对菌株进行了鉴定,并对其基本生物学特性进行了研究。结果表明:菌株KL-1对于七种病原菌极细链格孢菌、黑曲霉、青霉菌、拟康宁木霉、盐生枝孢霉、子囊菌和胶孢炭疽菌均有明显的抑制作用,其中对黑曲霉的抑制率最大为81%,对其他六种病原菌抑制率也均高于70%,对尖侧多隔孢霉的抑制率为0,说明菌株KL-1可以抑制番茄多种常见病原真菌,具有开发为生防制剂的潜力。经鉴定KL-1为解淀粉芽孢杆菌(Bacillus amyloliquefaciens);该菌培养9 h内生长最旺盛,最适生长pH为7.0,最适生长温度为37℃。     

Cloning,heterologous expression,and enzymatic characterization of a thermostable glucoamylase from Talaromyces emersonii

The gene encoding a thermostable glucoamylase from Talaromyces emersonii was cloned and, subsequently, heterologously expressed in Aspergillus niger. This glucoamylase gene encodes a 618 amino acid long protein with a calculated molecular weight of 62,827Da. T. emersonii glucoamylase fall into glucoside hydrolase family 15, showing approximately 60% sequence similarity to glucoamylase from A. niger. The expressed enzyme shows high specific activity towards maltose, isomaltose, and maltoheptaose, having 3-6-fold elevated k(cat) compared to A. niger glucoamylase. T. emersonii glucoamylase showed significantly improved thermostability with a half life of 48h at 65 degrees C in 30% (w/v) glucose, compared to 10h for glucoamylase from A. niger. The ability of the glucoamylase to hydrolyse amylopectin at 65 degrees C is improved compared to A. niger glucoamylase, giving a significant higher final glucose yield at elevated temperatures. The increased thermal stability is thus reflected in the industrial performance, allowing T. emersonii glucoamylase to operate at a temperature higher than the A. niger enzyme.     

Evaluation of Gliocladium roseum Against Wood-Degrading Fungi in vitro and on Major Canadian Wood Species

The protection of wood from fungal stain using biological agents has considerable potential for reducing discoloration of freshly sawn logs and lumber, while decreasing fungicide use. A number of biocontrol candidates have been reported worldwide, and Gliocladium roseum is one of such microorganisms. In this study, the bio-activity of G. roseum was investigated against different wood-degrading fungi on agar plates and wafers of 12 major Canadian wood species. Of the four sap-staining fungi tested on agar plates, Ophiostoma piceae and Alternaria alternata showed greater sensitivity than Aureobasidium pullulans or Cladosporium sphaerospermum to G. roseum . On wood wafers, a spore suspension of G. roseum (1 10 6 spores/ ml) provided satisfactory protection of wood from stain on western hemlock ( Tsuga heterophylla ), white spruce ( Picea glauca ), amabilis fir ( Abies amabilis ), balsam fir ( Abies balsamea ) and jack pine ( Pinus banksiana ). The antagonist also restricted the development of moulds and stain on black spruce ( Picea mariana ), lodgepole pine ( Pinus contorta ) and white pine ( Pinus strobus ), but did not protect Douglas fir ( Pseudotsuga menziesii ), red pine ( Pinus resinosa ), white birch ( Betula papyrifera ) and trembling aspen ( Populus tremuloides ). Logs of black spruce and jack pine treated with G. roseum were much less stained than untreated ones after a 4-month period of summer storage in the field. In an anti-decay test, no significant difference was found for weight loss between wood blocks treated with G. roseum and untreated samples. Application of G. roseum with low levels of an anti-sap stain chemical (NP-1) to wood wafers simultaneously did not produce a noticeable improvement in wood protection against stain compared with the chemical treatment alone.     

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.     

Gallotannin hydrolysis by immobilized fungal mycelia in a packed bed bioreactor.

Hydrolysis of gallotannin to gallic acid by immobilized mycelia of Aspergillus niger MTCC 282, Aspergillus fischerii MTCC 150, Fusarium solani MTCC 350 and Trichoderma viride MTCC 167 in a packed bed bioreactor was studied. Fungal mycelia preinduced with 5 g L-1 gallotannin were immobilized in calcium alginate gel (1.5%) and the resultant beads were packed in a column to a bed volume of 175 mm3. Gallotannin dissolved in distilled water was passed through the column and the eluate was recycled after adjusting pH to 6 with ammonium hydroxide (10%). Maximum hydrolysis of gallotannin was recorded by immobilized mycelia of F. solani and T. viride at 35 degrees and 45 degrees C after 175 and 60 min of residency period respectively. Optimum substrate concentration required for maximum hydrolysis was 10 g L-1 at pH 5 for both the fungi. Immobilized mycelia of A. niger and A. fischerii revealed maximum operational stability. Loss of activity after eighth run was in the order of-A. niger (no loss), A. fischerii (7.5%), F. solani (18%) and T. viride (18%). Stability in terms of retention of enzyme activity after 150 days of storage at 4 degrees C was A. niger (58%), A. fischerii (26.8%), F. solani (83%) and T. viride (85.1%).     

Survey on the mycoflora of Egyptian wheat grains and their lemmae and paleae

The plating technique has been used to study the fungus floras of covered and uncovered wheat grains and their lemmae and paleae on glucose-cellulose and 40% sucrose-Czapek's agar at 28° C. Seventy-two species and 28 genera were collected from the three microhabitats on the three types of media. On glucose-Czapek's agar the most frequent species were Aspergillus niger, A. flavus, A. terreus, A. nidulans, Alternaria alternata, Cladosporium herbarum and Fusarium Oxysporum. On cellulose and 40% sucrose-Czapek's agar, the composition of fungal floras of the three substrates and the frequency of prevalence of the individual fungi were basically similar to those obtained on glucose agar, but the frequency of some species was promoted or decreased.     

Extracellular proteinases of filamentous fungi as potential markers of phytopathogenesis

The presence of proteins in the culture liquid of filamentous fungi under study was found to induce the secretion of proteinases. The inhibitory analysis of the major extracellular proteinases of the saprotrophic fungus Trichoderma harzianum and the phytopathogenic fungus Alternaria alternata showed that they both belong to the group of serine proteinases. The substrate specificity of these proteinases and their sensitivity to inhibitors suggest that the enzyme of T. harzianum is a subtilisin-like proteinase and the enzyme of A. alternata is a trypsin-like proteinase. This difference between the proteinases may reflect the physiological difference between their producers (saprotroph and phytopathogen).     

Isolation of fungi in Musca domestica Linnaeus, 1758 (Diptera: Muscidae) captured at two natural breeding grounds in the municipality of Serop dica,Rio de Janeiro,Brazil

The objective of this study was to isolate and identify fungal species found in natural association with adults of Musca domestica. The adult insects were collected from two natural breeding grounds: hog pens and an urban sanitary landfill. The isolated fungi were identified as: Aspergillus flavus (23.8%), A. niger var. niger (14.4%), Penicillium corylophilum (21.4%), P. fellutanum (11.9%), Cladosporium cladosporoides (4.7%), Fusarium sp. (4.7%), Alternaria alternata (11.9%), Curvularia brachyspora (2.4%), Mycelia sterilia (2.4%) and the Mucorales order (2.4%).     

Molecular cloning and characterization of the fructooligosaccharide-producing beta-fructofuranosidase gene from Aspergillus niger ATCC 20611

The fopA gene encoding a fructooligosaccharide-producing beta-fructofuranosidase was isolated from Aspergillus niger ATCC 20611. The primary structure deduced from the nucleotide sequence showed considerable similarity to those of two other beta-fructofuranosidases from A. niger, but the fopA gene product had several amino acid insertions and an extra C-terminal polypeptide consisting of 38 amino acids that could not be found in the two others. We could successfully express the fopA gene in S. cerevisiae and the fopA gene product obtained from the culture supernatant of the S. cerevisiae transformant had similar characteristics to the beta-fructofuranosidase purified from A. niger ATCC 20611. However, we could not detect any beta-fructofuranosidase activity in either the culture supernatant or cell lysate when the C-terminal truncated fopA gene product by 38 amino acids was used to transform S. cerevisiae. In western analysis of those samples, there was no protein product that is cross-reacted with anti-beta-fructofuranosidase antibody. These results suggested that the C-terminal region of the fopA gene product consisting of 38 amino acids was essential for the enzyme production.