• The Korean Journal of Mycology
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• v.14 no.2
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• pp.141-148
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• 1986

The optimal conditions for high yields of mycelial protoplasts from P. cornucopiae were established. The concentraion of enzyme system containing Novozym 234, ${\beta}-D-glucanase$ and ${\beta}-glucuronidase$ was $5mg\;ml^{-1}$ each. The osmotic stabilizer most effective for protoplast isolation was O.6 M sucrose. The optimal reaction time of mycelium with the lytic mixture was 90 min in a shaking condition at 120 strokes $min^{-1}$. When the myelium of P. cornucopiae was cultured for 4 days on mushroom complete medium at $28^{\circ}C$, the formation of protoplast was effective. When the pH of the digestion mixture with O.6 M sucrose as stabilizer varied between pH 4.0 and 7.0, the production of protoplasts was effective in phosphate buffer (pH 6.2) and Na-maleate buffer (pH 5.0). Generally, phosphate buffer was more effective for protoplast isolation than Na-maleate buffer, but 0.6 M sucrose osmotic stabilizer without adjusting pH was most effective. Using these conditions, protoplasts from P. cornucopiae were obtained at a ratio $1{\times}10^7\;ml^{-1}$.

• The Plant Pathology Journal
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• v.28 no.3
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• pp.282-289
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• 2012

The root rot of pepper (Capsicum annuum L.) caused by Phytophthora capsici is one of the most important diseases affecting this crop worldwide. This work presents the evaluation of the capacity of Streptomyces griseus H7602 to protect pepper plants against Phytophthora capsici and establishes its role as a biocontrol agent. In this study, we isolated an actinomycete strain H7602 from rhizosphere soil, identified it as Streptomyces griseus by 16S rRNA analysis and demonstrated its antifungal activity against various plant pathogens including P. capsici. H7602 produced lytic emzymes such as chitinase, ${\beta}$-1,3-glucanase, lipase and protease. In addition, crude extract from H7602 also exhibited destructive activity toward P. capsici hyphae. In the pot trial, results showed the protective effect of H7602 against pepper from P. capsici. Application of H7602 culture suspension reduced 47.35% of root mortality and enhanced growth of pepper plants for 56.37% in fresh root and 17.56% g in fresh shoot as compared to control, resulting in greater protection to pepper plants against P. capsici infestation. Additionally, the enzymatic activities, chitinase and ${\beta}$-1,3-glucanase, were higher in rhizosphere soil and roots of pepper plants treated with H7602 than other treated plants. Therefore, our results indicated a clear potential of S. griseus H7602 to be used for biocontrol of root rot disease caused by P. capsici in pepper.

• Korean Journal of Soil Science and Fertilizer
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• v.38 no.1
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• pp.25-31
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• 2005

A soil bacterium, Bacillus subtilis HJ927, exhibiting strong antagonistic property against pathogenic fungi was isolated from pepper fields infested with Phytophthora capsici. Pepper plants inoculated with P. capsici revealed severe root mortality while plants co-inoculated with B. subtilis HJ927 and P. capsici showed drastically reduced root mortality. Low molecular weight substances released by B. subtilis HJ927 mediated the plant protective effect. The anti-fungal compounds released by B. subtilis HJ927 were identified as 3-methylbutyric acid, 2-methylbutyric acid, and methyl 2-hydroxy, 3-phenylpropanoate by high-performance liquid chromatography and gas chromatography-mass spectrometry. In addition to these compounds, B. subtilis HJ927 also produced ${\beta}$-1,3-glucanase, a hydrolytic enzyme implicated in antifungal activity.

• Korean Journal of Food Science and Technology
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• v.39 no.3
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• pp.276-282
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• 2007

In this study, attempts were made to develop a garlic juice extraction method that would result in minimum changes in quality. Protopectinase and a mutienzyme containing cellulase, pectinesterase, ${\beta}-glucanase$, etc. were applied to garlic residue after first extraction, and the yields of garlic juice and the flavor component changes of the juices were investigated. Enzyme concentrations of 0.04, 0.08, and 0.12% which were based on pulp weight before extraction were added and allowed to hydrolyze for 30, 60, 90 and 120 min,. respectively. Increase in the garlic juice yield was observed according to the amount of enzyme added and the reaction time until reaching a maximum point. When 0.12% protopectinase was applied to the garlic residue for 90 min, the yield increased by 13.8%. Under the same conditions, when multienzyme was applied to the garlic residue, the yield increased by 14.5%, which was considered the maximum. The flavor compounds decreased when compared with the total GC peak area of garlic juice prepared without enzymes(control). The volatile flavor compounds in garlic juice prepared with 0.12% protopectinase for 60 min decreased by 6%. The free sugars profile of the garlic juice prepared with 0.12% protopectinase for 60 min was similar to that of the control. The type of enzyme used did not affect the free amino acid profile of the garlic juice. These results indicate that the optimum conditions for extraction of garlic juice are hydrolyzing the residue with 0.12% protopectinase for 60 min, extracting garlic juice from the hydrolyzed reside, and then combining the extracted juice with the first extraction.

• Korean Journal of Microbiology
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• v.52 no.4
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• pp.437-443
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• 2016

In this study Streptomyces strains were isolated from soils and their antifungal activities and involved mechanisms were investigated. Among over 400 isolates of actinomycetes, Streptomyces costaricanus HR391 was selected as a potential antagonist to control several plant-pathogenic fungi. S. costaricanus HR391 inhibited mycelial growth of Fusarium oxysporum f. sp. raphani, F. oxysporum f. sp. niveum, F. oxysporum f. sp. lycopersici, and Rhizoctonia solani by 26.5, 26.2, 21.2, and 23.8%, respectively compared to those of uninoculated control after 7-day incubation on PDB medium. S. costaricanus HR391 produced $89{\mu}M$ of siderphore, and showed fungal cell wall-degrading activity including $0.46{\mu}mol/min/mg$ of chitinase and $0.83{\mu}mol/min/mg$ of ${\beta}$-1,3 glucanase. S. costaricanus HR391 secreted 87.49 mg/L of rhamnolipid, and produced 9.49 mg/L and 4.3 mM of lipopeptide, iturin A and surfactin, respectively, all they are membrane-disrupting biosurfactants. It also produced antimicrobial peptide and antibiotics phenazine. In addition to antifungal substances, S. costaricanus HR391 secreted plant growth-promoting phytohormones, zeatin, gibberellins and IAA. These results suggest that S. costaricanus HR391 may be utilized as an environment-friendly biocontrol agent against some important pathogenic fungi.

• Korean Journal of Soil Science and Fertilizer
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• v.43 no.4
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• pp.490-497
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• 2010

A chitinolytic bacterium having a strong antagonistic activity against various pathogens including Phytophtora capsici was isolated from rhizosphere soil, and identified as Lysobacter enzymogenes (named as LE429) based on 16S rRNA gene sequence analysis. This strain produced a number of substances such as chitinase, ${\beta}-1$, 3-glucanase, lipase, protease, gelatinase and an antibiotic compound. This antibiotic compound was purified by diaion HP-20, sephadex LH-20 column chromatography and HPLC. The purified compound was identified as phenylacetic acid by gas chromatography-electron ionization (GC-EI) and gas chromatography-chemical ionization (GC-CI) mass spectrometry. In field experiment, pepper plants were treated by the strain LE429 culture (CB), neem oil solution (NO), combination (CB+NO) or control (CON). Plant height and number of branches, flowers and pods of pepper plant in CB treatment were generally highest, and followed by CB+NO, CON and NO. The fungal pathogens were strongly inhibited, while several insect pests were discovered in CB treatment. Any insect pests were not found, while all fungal pathogens tested were not suppressed in NO treatment. However, in CB+NO treatment, non incidence of fungal pathogens and insect pests were found. The strain LE429 producing secondary metabolites with neem oil should be a potential agent to control fungal diseases and insect pests.

• Asian-Australasian Journal of Animal Sciences
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• v.29 no.1
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• pp.126-133
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• 2016

A gene from Actinomyces sp. Korean native goat (KNG) 40 that encodes an endo-${\beta}$-1,4-glucanase, EG1, was cloned and expressed in Escherichia coli (E. coli) $DH5{\alpha}$. Recombinant plasmid DNA from a positive clone with a 3.2 kb insert hydrolyzing carboxyl methyl-cellulose (CMC) was designated as pDS3. The entire nucleotide sequence was determined, and an open-reading frame (ORF) was deduced. The ORF encodes a polypeptide of 684 amino acids. The recombinant EG1 produced in E. coli $DH5{\alpha}$ harboring pDS3 was purified in one step using affinity chromatography on crystalline cellulose and characterized. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis/zymogram analysis of the purified enzyme revealed two protein bands of 57.1 and 54.1 kDa. The amino terminal sequences of these two bands matched those of the deduced ones, starting from residue 166 and 208, respectively. Putative signal sequences, a Shine.Dalgarno-type ribosomal binding site, and promoter sequences related to the consensus sequences were deduced. EG1 has a typical tripartite structure of cellulase, a catalytic domain, a serine-rich linker region, and a cellulose-binding domain. The optimal temperature for the activity of the purified enzyme was $55^{\circ}C$, but it retained over 90% of maximum activity in a broad temperature range ($40^{\circ}C$ to $60^{\circ}C$). The optimal pH for the enzyme activity was 6.0. Kinetic parameters, $K_m$ and $V_{max}$ of rEG1 were 0.39% CMC and 143 U/mg, respectively.

• Journal of the Korean Society of Food Science and Nutrition
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• v.14 no.4
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• pp.401-408
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• 1985

The yeast cell wall lytic action of the alkaline AL-protease, which was found out of the crude Zymolyase that a kind of yeast cell wall lytic $endo-{\beta}-1$, 3-glucanase produced from Arthrobacter luteus, was investigated with the viable cells of S. sake and it's cell wall preparation. AL-protease on the lysis of the viable yeast cells showed very low activities with the alone, but the lytic activities were highly increased with the combination of AL-protease and Zymolyase. On the stepwise treatment of the viable yeast cells with AL-protease and Zymolyase, the cells were lysed highly only by the course having a treatment with Zymolyase after pretreatment with AL-protease. Thus synergistic action of AL-protease was not observed with any some commercial enzymes, known as a type of alkaline and serine protease such as AL-protease, and was also found to be affected greatly by the culture conditions and species of the yeast tested. AL-protease caused the release of some peptide and a lot of sugar from the cell wall preparation, but could not lysed the cell wall more than 66%. Whereas Zymolyase could lysed the cell walls almost completely with alone. On the basis of these results, the synergistic action of AL-protease on the lysis of S. sake cells is hypothesized that at first AL-protease bind to the yeast cell surface layer consisting of mannan and protein, and then changes their conformation to facilitate the penetration of Zymolyase from the outside to the inside framework layer constituted of alkali insoluble ${\beta}-1,\;3-glucan$.

• The Korean Journal of Mycology
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• v.23 no.2 s.73
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• pp.129-138
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• 1995

DNA fragment being able to restore in vitro activity of ${\beta}-1,3-glucan$ synthase was cloned by transformation of the Saccharomyces cerevisiae LP353 mutant strain with genomic library constructed in the YCp50. For the selection of transformants which showed no detectable phenotype linked to recovery of the defect in ${\beta}-1,3-glucan$ synthase activity, the colony autoradiography was succesfully applied. The restriction map of the cloned DNA fragment, which is 8.5-kb in length, was constructed. Both the YEplac195 and the YCp50 carrying the 8.5-kb fragment increased ${\beta}-1,3-glucan$ synthase activity of LP353 by two fold. Neither the YEplac195 nor the YCp50 carrying the 8.5-kb DNA fragment, however, complemented the temperature-dependent osmotic sensitivity which is another distinctive phenotype of LP353. Subcloning experiments indicated that a functional region was located in 4.8-kb BglII-KpnI fragment. The 4.8-kb fragment was also able to increase the level of ${\beta}-1,3-glucan$ content in cell wall as well as the resistance of cells to cell wall lytic enzyme, ${\beta}-1,3-glucanase$. The growth rate of the LP353 with 4.8-kb fragment was almost same as that of wild type strain in liquid medium with 1.2 M sorbitol at nonpermissive temperature. Taken these results together, the 4.8-kb fragment seemed to contain the BGS2 gene for ${\beta}-1,3-glucan$ synthase activity in yeast S. cerevisiae.

• Microbiology and Biotechnology Letters
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• v.43 no.1
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• pp.56-64
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• 2015

It has been known for some time to the wine industry that non-Saccharomyces yeasts play an important role in increasing volatile components through the secretion of extracellular enzymes. The objective of this study was to investigate what types of enzymes are produced by 1,007 non-Saccharomyces yeast strains isolated from Korean fermented foods. Among 1,007 yeast strains, the 566, 45 and 401 strains displayed β-glucosidase, glucanase and protease activity, respectively. In addition, the 563 and 610 strains possessed tolerances against cerulenin and TFL, and the 307 strain was tolerant to 15% ethanol. Yeasts producing harmful biogenic amines and hydrogen sulfide were excluded from further study, and eventually 12 yeast strains belonging to the genera Wickerhamomyces, Hanseniaspora, Pichia, Saccharomyces were identified, based on the 26S rRNA gene sequences. Among the 12 strains, the 9 and 5 strains possessed glucose and ethanol tolerance, respectively. Yeasts belonging to the genus Saccharomyces produced more than 8% alcohol, but non-Saccharomyces yeasts produced only 3% alcohol.