• Journal of Microbiology and Biotechnology
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• v.21 no.8
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• pp.808-817
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• 2011

Because of its elevated cellulolytic activity, the filamentous fungus Trichoderma harzianum has a considerable potential in biomass hydrolysis applications. Trichoderma harzianum cellobiohydrolase I (ThCBHI), an exoglucanase, is an important enzyme in the process of cellulose degradation. Here, we report an easy single-step ion-exchange chromatographic method for purification of ThCBHI and its initial biophysical and biochemical characterization. The ThCBHI produced by induction with microcrystalline cellulose under submerged fermentation was purified on DEAE-Sephadex A-50 media and its identity was confirmed by mass spectrometry. The ThCBHI biochemical characterization showed that the protein has a molecular mass of 66 kDa and pI of 5.23. As confirmed by smallangle X-ray scattering (SAXS), both full-length ThCBHI and its catalytic core domain (CCD) obtained by digestion with papain are monomeric in solution. Secondary structure analysis of ThCBHI by circular dichroism revealed ${\alpha}$- helices and ${\beta}$-strands contents in the 28% and 38% range, respectively. The intrinsic fluorescence emission maximum of 337 nm was accounted for as different degrees of exposure of ThCBHI tryptophan residues to water. Moreover, ThCBHI displayed maximum activity at pH 5.0 and temperature of $50^{\circ}C$ with specific activities against Avicel and p-nitrophenyl-${\beta}$-D-cellobioside of 1.25 U/mg and 1.53 U/mg, respectively.

• KSBB Journal
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• v.11 no.1
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• pp.22-29
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• 1996

Immobilized bioprocess was carried out for continuous production of cyclosporin A (CyA) produced intracellularly as a secondary metabolite by a filamentous fungus, Tolypocladium inflatum. Immobilization procedure for entrapping conidiospores of the producer was significantly simplified by use of a modified immobilization technique. A newly-designed immobilized perfusion reactor system (IPRS) showed good process benefits as demonstrated by the role of the high density immobilized cells as an efficient biomass generator, continuously supplying highly active CyA-producing free cells (1.0g/$\ell$/hr) even at very high dilution rate ($0.1hr^{-1}$). IPRS bioprocess was possible since efficient decantor system developed in our laboratory separated the sloughed-off free cells from the immobilized biomass effectively, thus overcoming wash-out phenomenon frequently encountered in continuous free cell cultures. Furthermore the released-free cells remaining in the bulk solution did not appear to cause substrate mass transfer limitation which was often experienced in suspended mycelial fungal cell fermentations. The primary reason for this was that the suspension broth of the IPRS mainly consisted of roundshaped short mycelial fragments and conidiospores, still remaining Newtonian even at high cell density. In parallel with IPRS bioprocess development, other key factors to be considered necessarily for significant increase in CyA productivity would be strain improvement and medium optimization for the immobilized cells.

• 한국생물공학회:학술대회논문집
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• 2002.04a
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• pp.441-444
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• 2002

This study was targeted to develope a microbial consortium having high cellulase production. A filamentous fungus, strain FB01, isolated from a compost showed high ${\beta}-glucosidase$ activity especially. The strain FBOl was co-cultured with Trichoderma viride to enhance the productivity of ${\beta}-glucosidase$, changing inoculation time of one strain (FB01). The microbial consortium prepared showed the higher cellulytic enzyme production than T. viride well-known. The maximal enzyme production was obtained when the microbial consortium was cultured at $30^{\circ}C$ and pH 6.0 for 10days and the activities of CMCase, ${\beta}-glucosidase$, and avicelase were 2.0, 0.8, and 0.2 U/mL, respectively. These enzyme activities were 2, 4, and 2 times as high as those of CMCase, ${\beta}-glucosidase$, avicelase from T. viride, respectively, indicating that a synergistic interaction appeared between T viride and strain FB01. The serial subcultures by pH control increased ${\beta}-glucosidase$ production about 3.2 times. Also, enzyme production using rice-straw as a carbon source showed that the activities of CMCase, ${\beta}-glucosidase$, and avicelase were 3.69, 0.76, 0.17 U/mL, respectively, and ${\beta}-glucosidase$ activity was 1.5 times higher than that of T. viride. Consequently, microbial consortium showed the considerabely enhanced production of the cellullolytic enzymes, such as CMCase, ${\beta}-glucosidase$, and avicelase compared those of T. viride, and a favorable stability for the enzyme production even in the serial subcultures.

• The Korean Journal of Mycology
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• v.15 no.3
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• pp.187-195
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• 1987

Two kinds of heat treatment method for the selective isolation of soil fungi to eliminate the commonest fungi and also to examine the vertical and seasonal distributions of the fungal population were applied to soil samples from two plots around Seoul area. The incubation method at $42^{\circ}C$ and heat treatment at $70^{\circ}C$ were used in this experiment. In the incubation method, the almost all the fungi isolated from two plots were mesophile, while the thermotolerant fungi was Aspergillus fumigatus and thermophilic fungi were Sporotrichum thermophile and Malbranchea pulchella var. sulfrea. The most dominant species isolated by this method was A. fumigatus. Nine genera and fourteen species were isolated from the two plots, and S. thermophile, Talaromyces ucrainicus,Malbranchea pulchella var. sulfrea were new to Korea. From the selection method by heat treatment at $70^{\circ}C$, ten genera and twenty species were isolated. Among these, the most fungi were also mesophile and thermotolerant fungus was A. fumigatus. The most dominant species isolated by this method was T. stipitatus, Talaromyces helicus var. major, Emericella nidulans var. nidulans, Chaetomium subspirale and Neosartorya fisheri var. fisheri were new to Korea. From the two isolation methods, it was found that the total number of soil fungi and frequency of species appeared including dominant ones were the highest at the soils of upper layer while the lowest at the soils of lower layer in its vertical distribution.

• 한국균학회소식:학술대회논문집
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• 2014.10a
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• pp.13-13
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• 2014

Species belonging to the genus Fusarium are widely distributed and cause diseases in many plants. Isolation of fungal strains from air or cereals is necessary for disease forecasting, disease diagnosis, and population genetics [1]. Previously we showed that Fusarium species are resistant to toxoflavin produced by the bacterial rice pathogen Burkholderia glumae while other fungal genera are sensitive to the toxin, resulting in the development of a selective medium for Fusarium species using toxoflavin [2]. In this study, we have tried to elucidate the resistant mechanism of F. graminearum against toxoflavin and interaction between the two pathogens in nature. To test whether B. glumae affects the development of F. graminearum, the wild-type F. graminearum strains were incubated with either the bacterial strain or supernatant of the bacterial culture. Both conditions increased the conidial production five times more than when the fungus was incubated alone. While co-incubation resulted in dramatic increase of conidial production, conidia germination delayed by either the bacterial strain or supernatant. These results suggest that certain factors produced by B. glumae induce conidial production and delay conidial germination in F. graminearum. To identify genes related to toxoflavin resistance in F. graminearum, we screened the transcriptional factor mutant library previously generated in F. graminearum [3] and identified one mutant that is sensitive to toxoflavin. We analyzed transcriptomes of the wild-type strain and the mutant strain under either absence or presence of toxoflavin through RNAseq. Expression level of total genes of 13,820 was measured by reads per kilobase per million mapped reads (RPKM). Under the criteria with more than two-fold changes, 1,440 genes were upregulated and 1,267 genes were down-regulated in wild-type strain than mutant strain in response to toxoflavin treatment. A comparison of gene expression profiling between the wild type and mutant through gene ontology analysis showed that genes related to metabolic process and oxidation-reduction process were highly enriched in the mutant strain. The data analyses will focus on elucidating the resistance mechanism of F. graminearum against toxoflavin and the interaction between the two pathogens in rice. Further evolutionary history will be traced through figuring out the gene function in populations and in other filamentous fungi.

• The Korean Journal of Mycology
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• v.41 no.3
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• pp.192-196
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• 2013

Fungal development is largely affected by many environmental factors. In a model filamentous fungus Aspergillus nidulans, asexual development is promoted by exposure of light, presence of salt and non-fermentable sugars. In other hand, sexual development is largely induced by absence of light, fermentable sugars and hypoxic condition. Also, some important genes including veA and nsdD play positive roles in activating sexual development. Here, we reported that the effect of high concentration of $CO_2$ on developmental decision in A. nidulans. When wild-type $veA^+$ strain was cultured in normal condition, sexual and asexual development occurred in balanced manner. However, high concentration of $CO_2$ (~5%) strongly activated sexual development and inhibited asexual development. Furthermore, this $CO_2$ effect was controlled by the veA or nsdD gene. High $CO_2$ culture of $veA^-$ or $nsdD^-$ mutant didn't activate sexual development, suggesting that the activation of sexual development induced by high $CO_2$ cannot overcome the genetic requirement of sexual development such as veA or nsdD. Since 5% $CO_2$ is an important condition for human pathogenic fungi for surviving and adapting in human body, this developmental pattern of A. nidulans affected by $CO_2$ concentration may provide interesting clues for comparative study with human fungal pathogens including Aspergillus fumigatus.