• Korean Journal of Soil Science and Fertilizer
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• v.39 no.4
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• pp.185-194
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• 2006

A fungal strain of Trichoderma having strong chitinolytic activity was isolated from field soil enriched with crabshell for several years. Based on 5.8S rRNA, partial 18S, 28S rRNA genes, ITS1, ITS2 sequence analysis and morphological characteristics, the fungus was identified as Trichoderma asperellum and named as Trichoderma asperellum T-5 (TaT-5). The fungus released lytic enzymes such as chitinase and ${\beta}$-1, 3-glucanse, and produced six antifungal substances in chitin broth medium. To demonstrate the protective effect of TaT-5 against damping-off in cucumber plant caused by Rhizoctonia solani, TaT-5 culture broth (TA), chitin medium (CM) and distilled water (DW) were applied to each pot at 10 days after sowing, respectively. Then, the homogenized hyphae of R. solani were infected to each pot at 1 week after TaT-5 inoculation. During experimental period, fresh weight of shoot and root in cucumber plant more increased at TA treatment compared to other treatments. PR-proteins (${\beta}$-1, 3-glucanase and chitinase) activities in cucumber leaves markedly increased at CM and DW treatments, but the activity slightly increased and then decreased at TA treatment at 3 days after infection of R. solani. The activity of PR-proteins activities in cucumber roots at all treatments decreased with time where the degree of decrement was more alleviated at TA treatment than CM and DW. These results suggest that the lytic enzymes (chitinase and ${\beta}$-1, 3-glucanse) and antifungal substances produced by TaT-5 can reduce the pathogenic attack by R. solani in cucumber plants.

• Korean Journal of Microbiology
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• v.40 no.1
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• pp.54-59
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• 2004

$\beta$-Glucan has been efficiently produced with higher yield by the optimization of liquid cultivation conditions. The optimal composition of medium for batch culture was 5% (w/v) of glucose as a carbon source, 0.5% (w/v) of yeast and 0.5% (w/v) of malt extract as a nitrogen source, 0.1% (w/v) of $KH_2PO_4$ and 0.05% (w/v) $MgSO_4{\cdot}7H_2O$, which had been the base medium for determination of other conditions. The set-up conditions are pH 5.0, $28^{\circ}C$, 1 vvm for aeration and 300 rpm for agitation. In order to minimize the inhibition effect of glucose on the initial growth of mycelia and to maximize the production of extracellular $\beta$-glucan, we have reduced the initial glucose feed to 4% and added 2nd feed at the point of 70 hr from the initial feed. The 2nd feed was composed of glucose 3%, yeast extract 0.1 % and malt extract 0.1 %. It improved the $\beta$-glucan yield upto 5.2 g/L in comparison with 2.8 g/L resulted from batch cultivation. Moreover, the serial treatment of a cell wall lytic enzyme and bromelain to the mycelia was effective for extraction of the cell wall bound $\beta$-glucan. The yield of $\beta$-glucan extraction by the enzyme treatment was 3.5 g/L, which was almost 4 times higher than that by hot-water extraction.

• Journal of the Korea Academia-Industrial cooperation Society
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• v.9 no.2
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• pp.550-555
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• 2008

An efficient production method of spirulina extract was developed by enzymatic treatment using proteolytic enzymes. The suitable dosage of Tunicase, a cell lytic enzyme, was used to be 2.0% (w/w). To maximize solid recovery and spirulina extraction (SE) index, which indicates nucleic acid-related substances content, the dosage of Alcalase, commercially available pretense, was found to be 1.0% (w/w). By simultaneous treatments using optimal dosages of Tunicase and Alcalase, the highest SE index and solid recovery were obtained. The SE index and solid recovery of simultaneous treatments were notably enhanced by 100% ($11.4%\;{\rightarrow}\;22.8%$) and 56% ($45.2%\;{\rightarrow}\;70.7%$), respectively, than those of the non-treated extracts.

• Korean Journal Plant Pathology
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• v.3 no.2
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• pp.124-130
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• 1987

The optimum conditions for protoplast formation and regeneration from Pyricularia oryzae Cav. were selected as follows. As a basic solution, 0.02M potassium phosphate buffer solution plus 0.6M KCl adjusted to pH 5.2 with 1N HCl was used. A mixture of enzyme combinations with 20mg Cellulase R-l0/ml, 5mg Macerozyme R-l0/ml and l0mg Driselase/ml used as a lytic enzyme showed better lytie effect than any single enzyme treatment for protoplast formation. Two-day-old mycelia of P. oryzae grown in the mixture of three lytie enzyme solution at $30^{\circ}C$ for 3 hr showed best condition for protoplasts formation. For regeneration from the protoplasts of P. oryzae, potato dextrose agar containing 0.02M potassium phosphate plus 0.6M KCl used as a stabilizer was best for regeneration medium.

• Microbiology and Biotechnology Letters
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• v.10 no.4
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• pp.245-252
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• 1982

The enzymes lytic to red yeast cell wall, which were produced by Penicillium lilacinum ATCC 36010 and Bacillus pumilus No 41, hydrolyzed an extracellular mannan from Rhodotonla glutinis IFO 0695. mannan was arranged with $\beta$-1,3 and $\beta$-1,4 linkages alternatively. Using this mannan, substrate specificities of these enzymes were investigated. The one from Penicillium lilacinum was an unique mannanase which hydrolyzed $\beta$-1,3 mannoside bond and the other from B. pumilus was a new type of mannanase which cleaved $\beta$-1,4 mannoside bond with requirement of the existence of $\beta$-1,3 linkage on the reducing side. Both enzymes released two kinds of oligosaccharide from mannan, respectively. However, the enzyme from Pen lilacinum produced tetrasaccharide and disaccharide and one of them, tetrasaccharide, was hydrolyzed to disaccharide further. The one from B. pumilus released tetrasaccharide and hexasaccharide from mannan finally.

• Journal of Microbiology and Biotechnology
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• v.24 no.6
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• pp.803-811
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• 2014

Concerns over drug-resistant bacteria have stimulated interest in developing alternative methods to control bacterial infections. Endolysin, a phage-encoded enzyme that breaks down bacterial peptidoglycan at the terminal stage of the phage reproduction cycle, is reported to be effective for the control of bacterial pathogenic bacteria. Bioinformatic analysis of the SPN9CC bacteriophage genome revealed a gene that encodes an endolysin with a domain structure similar to those of the endolysins produced by the P1 and P22 coliphages. The SPN9CC endolysin was purified with a C-terminal oligo-histidine tag. The endolysin was relatively stable and active over a broad temperature range (from $24^{\circ}C$ to $65^{\circ}C$). It showed maximal activity at $50^{\circ}C$, and its optimum pH range was from pH 7.5 to 8.5. The SPN9CC endolysin showed antimicrobial activity against only gram-negative bacteria and functioned by cutting the glycosidic bond of peptidoglycan. Interestingly, the SPN9CC endolysin could lyse intact gram-negative bacteria in the absence of EDTA as an outer membrane permeabilizer. The exogenous lytic activity of the SPN9CC endolysin makes it a potential therapeutic agent against gram-negative bacteria.

• The Plant Pathology Journal
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• v.31 no.1
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• pp.50-60
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• 2015

The use of novel isolates of Trichoderma with efficient antagonistic capacity against Fusarium oxysporum f. sp. lycopersici (FOL) is a promising alternative strategy to pesticides for tomato wilt management. We evaluated the antagonistic activity of 30 isolates of T. asperellum against 4 different isolates of FOL. The production of extracellular cell wall degrading enzymes of the antagonistic isolates was also measured. The random amplified polymorphic DNA (RAPD) method was applied to assess the genetic variability among the T. asperellum isolates. All of the T. asperellum isolates significantly reduced the mycelial growth of FOL isolates but the amount of growth reduction varied significantly as well. There was a correlation between the antagonistic capacity of T. asperellum isolates towards FOL and their lytic enzyme production. Isolates showing high levels of chitinase and ${\beta}$-1,3-glucanase activities strongly inhibited the growth of FOL isolates. RAPD analysis showed a high level of genetic variation among T. asperellum isolates. The UPGMA dendrogram revealed that T. asperellum isolates could not be grouped by their antagonistic behavior or lytic enzymes production. Six isolates of T. asperellum were highly antagonistic towards FOL and potentially could be used in commercial agriculture to control tomato wilt. Our results are consistent with the conclusion that understanding the genetic variation within Trichoderma isolates and their biochemical capabilities are required for the selection of effective indigenous fungal strains for the use as biocontrol agents.

• Korean Journal of Microbiology
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• v.33 no.4
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• pp.242-246
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• 1997

The protein profile of Synechococcus sp. cyanophage was investigated employing SDS-PAGE. The phage appears to be composed of two major proteins of 97 and 52 kDa and at least seven minor proteins of 70, 65, 60, 40, 35, 28, and 6 kDa. It seems that each subunit is combined to form a multimer although any disulfide bond does not exist in the phage structure. Lytic activity of the phage particle against cell wall was detected around the 52 kDa on renaturing SDS-PAGE using heat-killed Micrococcus luteus cells as substrate. The activity has the optimal pH between 9 and 10, and slightly inhibited by EDTA.

• Asian-Australasian Journal of Animal Sciences
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• v.27 no.4
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• pp.574-579
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• 2014

A lysozyme gene from breast of Tibetan sheep was successfully expressed by secretion using a-factor signal sequence in the methylotrophic yeast, Pichia pastoris GS115. An expression yield and specific activity greater than 500 mg/L and 4,000 U/mg was obtained. Results at optimal pH and temperature showed recombinant lysozyme has higher lytic activity at pH 6.5 and $45^{\circ}C$. This study demonstrates the successful expression of recombinant lysozyme using the eukaryotic host organism P. pastoris paving the way for protein engineering. Additionally, this study shows the feasibility of subsequent industrial manufacture of the enzyme with this expression system together with a high purity scheme for easy high-yield purification.

• Journal of Life Science
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• v.13 no.2
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• pp.143-149
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• 2003

This experiment was undertaken to investigate proper conditions for protoplast isolation and regeneration from the mycelia of Lyophyllum ulmnrium. Protoplast isolation and regeneration are influenced by a variety of factors such as enzyme, osmotic stabilizer, reaction time and age of mycelia. A combination of Novozyme 234 (10mg/ml) and cellulase Onozuka R-10 (10 mg/ml) with 0.6 M $MgSO_4$ was most effective for isolation of the protoplasts. The optimum reaction time of the mycelia with the lytic enzymes was 3.5~4 hours at $28^{\circ}C$ in shaking condition at 120 strokes per min. High yield of the protoplasts were obtained from its 4~5 days old mycelia on complete agar media. Its protoplasts were regenerated to normal hyphae. Regeneration media with 0.6 M sucrose were proper for regeneration of the protoplasts. Their regeneration frequency on complete agar media was 2.3~2.7%.