• Bulletin of the Korean Chemical Society
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• v.28 no.12
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• pp.2303-2309
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• 2007

Expressed protein ligation (EPL) technique, joining recombinantly expressed proteins to polypeptides, has been widely adopted for addressing various biological questions and for drug discovery. However, joining two recombinant proteins together is sometimes difficult when proteins are expressed insoluble and unrefoldable, because ligation-active proteins via intein-fusion are obtainable when they are folded correctly. We overcame this limitation coexpressing target protein with additional methionine aminopeptidase (MAP) which enhances removal of the initiation methionine of recombinantly expressed protein. Our approach demonstrated that two domains of 46 kDa 5-Enolpyruvylshikimate-3-phosphate (EPSP) synthase, a target of herbicide glyphosate, were successfully joined by native chemical ligation, although its C-terminal domain was expressed as an inclusion body. The intein-fused N-terminal fragment of EPSP synthase (EPSPSN, residues 1-237) was expressed and the ligation-active thioester tagged N-terminal fragment (EPSPSN-thioester) was purified using a chitin affinity chromatography and mercapto-ethanesulphonate (MESNA) as intein thiolysis reagent. Its Cterminal fragment (EPSPSC, residues Met237-238CYS-427), expressed as an inclusion body, was prepared from an additional MAP-expressing strain. Protein ligation was initiated by mixing ~1 mM of EPSPSN-thioester with ~2 mM of EPSPSCCYS (residues 238CYS-427). Also we found that addition of 2% thiophenol increased the ligation efficiency via thiol exchange. The ligation efficiency was ~85%. The ligated full-length EPSP synthase was dissolved in 6 M GdHCl and refolded. Circular dichroism (CD) and enzyme activity assay of the purified protein showed that the ligated enzyme has distinct secondary structure and ~115% specific activity compared to those of wild-type EPSP synthase. This work demonstrates rare example of EPL between two recombinantly expressed proteins and also provides hands-on protein engineering protocol for large proteins.

• The Plant Pathology Journal
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• v.28 no.4
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• pp.439-445
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• 2012

The chitinase producing Lysobacter enzymogenes C-3 has previously been shown to suppress plant pathogens in vitro and in the field, but little is known of the regulation of chitinase production, or its role in antimicrobial activity and biocontrol. In this study, we isolated and characterized chitinase-defective mutants by screening the transposon mutants of L. enzymogenes C-3. These mutations disrupted genes involved in diverse functions: glucose-galactose transpoter (gluP), disulfide bond formation protein B (dsbB), Clp protease (clp), and polyamine synthase (speD). The chitinase production of the SpeD mutant was restored by the addition of exogenous spermidine or spermine to the bacterial cultures. The speD and clp mutants lost in vitro antifungal activities against plant fungal pathogens. However, the gluP and dsbB mutants showed similar antifungal activities to that of the wild-type. The growth of the mutants in nutrient rich conditions containing chitin was similar with that of the wild-type. However, growth of the speD and gluP mutants was defective in chitin minimal medium, but was observed no growth retardation in the clp and dsbB mutant on chitin minimal medium. In this study, we identified the four genes might be involved and play different role in the production of extracellular chitinase and antifungal activity in L. enzymogenes C-3.

• Mycobiology
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• v.46 no.2
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• pp.154-158
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• 2018

Bitter rot caused by the fungal genus Colletotrichum is a well-known, common disease of apple and causes significant yield loss. In 2013, six fungal strains were isolated from Fuji apple fruits exhibiting symptoms of bitter rot from Andong, Korea. These strains were identified as Colletotrichum fructicola and C. siamense based on morphological characteristics and multilocus sequence analysis of the internal transcribed spacer rDNA, actin, calmodulin, chitin synthase, and glyceraldehyde-3-phosphate dehydrogenase Pathogenicity tests confirmed the involvement of C. fructicola and C. siamense in the development of disease symptoms on apple fruits. This is the first report of C. fructicola and C. siamense causing bitter rot on apple fruit in Korea.

• Mycobiology
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• v.48 no.1
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• pp.75-79
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• 2020

Anthracnose is one of the major problems for cultivating many crops, including vegetables, fruits, and trees. It is a continual threat for fruits grower worldwide. Colletotrichum fructicola was isolated from Shine Muscat berries showing typical anthracnose symptom in Korea. It was identified as C. fructicola based on morphology, pathological signs and concatenated sequences of internal transcribed spacer region of rDNA, glyceraldehyde-3-phosphate dehydrogenase, β-tubulin-2, chitin synthase-1, calmodulin, and the Apn2-Mat1-2 intergenic spacer and partial mating type (Mat1-2) gene. To the best of our knowledge, this is the first report first report of anthracnose of Shine Muscat caused by C. fructicola in Korea.

• Mycobiology
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• v.48 no.4
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• pp.321-325
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• 2020

A Colletotrichum species was isolated from leaves of Cymbidium exhibiting symptoms of anthracnose. In this study, the isolates obtained were identified based on recent taxonomic approaches for the genus Colletotrichum. The identity of the causal pathogen was confirmed using morphological data and phylogenetic analysis of combined multi-gene dataset (internal transcribed spacer, glyceraldehyde 3-phosphate dehydrogenase, chitin synthase-1, actin, histone3, beta-tubulin, and calmodulin). Pathogenicity testing revealed that the isolates were pathogenic to Cymbidium. Based on these results, the fungal pathogen occurring on Cymbidium orchids was identified as Colletotrichum cymbidiicola, which is a newly recorded species in Korea.

• Journal of Chitin and Chitosan
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• v.22 no.3
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• pp.185-193
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• 2017

Red tide Heterosigma akashiwo (H. akashiwo), a microscopic alga of the class Raphidophyceae, causes extensive damage to all marine ecosystems. It is essential to reduce the damage to marine ecosystems for them to be used as a resource. In this study, we used organic solvent fractionation to obtain an ethyl acetate-methanol extract from H. akashiwo (HAEM80) and then evaluated its anti-inflammatory activity in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells and a zebrafish model. HAME80 markedly inhibited the production of nitric oxide (NO) and prostaglandin $E_2$ ($PGE_2$). It also down-regulated the protein expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) and decreased the secretion of interleukin-$1{\beta}$ ($IL-1{\beta}$) in LPS-stimulated RAW 264.7 cells. HAME80 reduced yolk edema and improved the survival rate of LPS-stimulated zebrafish embryos; in addition, the extract significantly reduced the production of ROS and NO and attenuated cell death in this model. Gas chromatography-mass spectrometry (GC-MS) of the extract was used to confirm the identity of peaks 1-20. Taken together, our data suggest that H. akashiwo is a beneficial anti-inflammatory agent.

• The Korean Journal of Mycology
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• v.47 no.4
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• pp.393-406
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• 2019

In August 2017, anthracnose symptoms were observed on the petioles and leaf veins of Korean radish (Rhaphanus sativus) in Hongcheon, Jeongseon, and Pyeongchang of the Gangwon province, Korea. Many grayish to dark-brown spots of 1-2 mm in diameter, appeared on the lower surface and leaf veins of the radish leaves. The spots gradually enlarged and coalesced to form dark-brown irregular lesions. Ten Colletotrichum isolates were obtained from the affected tissues of the Korean radish. Out of them, eight isolates were identified as C. higginsianum and two isolates were identified as C. truncatum based on morphological characteristics and multi-locus molecular phylogenetic analysis using the internal transcribed spacers and intervening 5.8S rDNA (ITS), partial beta-tubulin gene (TUB2), partial actin gene (ACT), and partial chitin synthase-1 gene (CHS1). The pathogenicity test was carried out on wounded and unwounded Korean radish (cv. Siraegimu and Osarimu), and Chinese cabbage (cv. Chuno and Smart) by inoculating with a spore suspension. All isolates except one C. truncatum isolate developed typical symptoms on both wounded and unwounded Korean radish. In Chinese cabbage, only the plants inoculated with C. higginsianum isolates developed symptoms regardless of the wound. This is the first report of anthracnose caused by C. truncatum on Korean radish in Korea.

• The Korean Journal of Pesticide Science
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• v.6 no.2
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• pp.105-109
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• 2002

Synthetic method of a new benzoylphenylurea insecticide, DBI-3204 [1-(2,6-Difluorobenzoyl)-3-(3,5-bistrifluoromethyl-2-chlorophenyl) urea] was established for manufactural production; 2,6-Difluorobenzoylisocyanate was synthesized by the reaction of 2,6-difluorobenzamide with oxalylchloride in 95% yields, the reaction of 3,5-bis(trifluoromethyl)aniline with N-chlorosuccinimde gave 3,5-bis (trifluoromethyl)-2-chloroaniline in 92% yields, and DBI-3204 was got from 2,6-difluorobenzoylisocyanate and 3,5-bis(trifluoromethyl)-2-chloroaniline in 90% yields. All the process was simplified by using the same reaction solvent, carbontetrachloride.

• Research in Plant Disease
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• v.27 no.2
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• pp.70-75
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• 2021

The abnormal brown sunken lesions were observed on cv. RubyS apple fruits in an orchard located in Gunwi, Gyeongbuk province, Korea. The primary observed symptoms such as small round sunken lesions and small black dots on the symptomatic area were different from the reported apple diseases. The affected apple fruits were sampled and subjected to isolation of the causal agent. Cultural and morphological characteristics of isolated fungal strain, designated KNUF-20GWA4, were similar to that of Colletotrichum spp. Based on multilocus sequence analyses using internal transcribed spacer regions and partial sequences of β-tubulin, glyceraldehyde-3-phosphate dehydrogenase, chitin synthase, and actin genes, strain KNUF-20GWA4 showed 99.2-100% similarities with C. aenigma ICMP 18608 and the isolate clustered together with several other strains of this species in the phylogenetic tree. To our knowledge, this is the first report of bitter rot on apple fruits caused by C. aenigma.

• The Korean Journal of Mycology
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• v.50 no.3
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• pp.235-241
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• 2022

Anthracnose crown rot (ACR) has been observed in greenhouses during the nursery and harvest seasons in Gangwon Province, Korea. Infected plants showed black leaf spot, dark sunken pink conidial masses on petioles, wilting, and eventually death. Five isolates were obtained from the lesions of strawberry plants and were identified as a Colletotrichum gloeosporioides species complex based on their cultural and morphological characteristics. Multilocus sequence analysis of actin, calmodulin, chitin synthase, glyceraldehyde-3-phophate dehydrogenase genes, and internal transcribed spacer rDNA regions showed that the isolates formed a monophyletic group with the type strain of C. siamense. Pathogenicity tests were performed on the isolate, and Koch's postulates were performed to verify the relationship between Colletotrichum sp. and the strawberry plant variety Seolhyang. The isolate was pathogenic to strawberry plants, which exhibited typical ACR symptoms. Based on morphological characteristics, pathogenicity, and DNA sequence analyses, the fungus isolated in Korea was identified as C. siamense. This is first time C. siamense has been confirmed in ever-bearing strawberry varieties in Korea.