• Proceedings of the Korean Society of Tobacco Science Conference
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• 1997.10a
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• pp.134-152
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• 1997

Plant viruses of tobacco including tobacco mosaic virus (TMV) and potato virus Y (PVY) cause severe economic losses in leaf-tobacco production. Cultural practices do not provide sufficient control against the viruses. Use of valuable resistant cultivars is most recommendable for the control of the viruses. However, conventional breeding programs are not always proper for the development of virus-resistant plants mostly owing to the frequent lack of genetic sources and introduction of their unwanted properties. Therefore, we tried to develop virus-resistant tobacco plants by transforming commercial tobacco cultivars, NC 82 and Burley 21, with coat protein (CP) or replicase (Nlb) genes of TMV and PVY necrosis strain (PVY-VN) with or without untranslated region (UTR) and with or without mutation. Each cDNA was cloned and inserted in plant expression vectors with 1 or 2 CaMV 35S promotors, and introduced into tobacco leaf tissues by Agrobacterium tumefaciens LBA 4404. Plants were regenerated in kanamycin-containing MS media. Regenerated plants were tested for resistance to TMV and PVY In these studies, we could obtain a TMV-resistant transgenic line transformed with TMV CP and 6 genetic lines with PVY-VN cDNAs out of 8 CP and replicase genes. In this presentation, resistance rates, verification of gene introduction in resistant plants, stability of resistance through generations, characteristics of viral multiplication and translocation in resistant plants, and resistance responses relative to inoculum potential and to various PVY strains will be shown. Yield and quality of leaf tobacco of a promising resistant tobacco line will be presented.

• Microbiology and Biotechnology Letters
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• v.38 no.1
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• pp.40-45
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• 2010

The gene encoding $\beta$-agarase (agaB) which hydrolyzes $\beta$-1,4 linkages of agarose from Zobellia galactanivorans was cloned and fused to Saccharomyces cerevisiae mating factor alpha-1 secretion signal ($MF{\alpha}1$), in which the transcription of $MF{\alpha}1$-AgaB was under the control of AOX1 (alcohol oxidase 1, methanol inducible) promoter. The constructed plasmid pPIC-AgaB (9 kb) was integrated into HIS4 gene locus of Pichia pastoris genome. Successful integration was confirmed by performing colony PCR. The transformed cells showed red halos around its colonies in methanol agar plate by adding iodine solution, indicating the active expression of agaB in P.pastoris. By SDS-PAGE and zymographic analysis, the molecular weight of $\beta$-agarase was estimated to be a 53 kDa and about 15% N-linked glycosylation was occurred. The activity of extracellular $\beta$-agarase reached 1.34, 1.42 and 1.53 units/mL by inducing 0.1, 0.5, and 1% methanol, respectively, at baffled flask culture of P.pastoris GS115/pPIC-AgaB for 48 hr. Most of the enzyme activity was found in the extacellular fraction and the secretion efficiency showed 98%. Thermostability of recombinant $\beta$-agarase was also increased by glycosylation.

• Korean Journal of Microbiology
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• v.49 no.2
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• pp.156-164
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• 2013

Actinomycetes produce diverse secondary metabolites which have the primary importance in medicine, agriculture and food production, and key to this is their ability to interact with other organisms in natural habitats. In this study, we have investigated the taxonomical and functional diversity of actinomycetes in fecal sample of rhinoceros beetle larvae (Allomyrina dichotoma L.) by using culture-dependent and -independent approaches. For the culture-independent approach, the community DNA was extracted from the sample and 16S rRNA genes of actinomycetes were amplified using actinomycetes-specific PCR primers. Thirty-seven clones were classified into 15 genera and 24 species of actinomycetes. For the culture-dependent approach, 53 strains were isolated from larval feces, of which 27 isolates were selected based on morphological characteristics. The isolates were classified into 4 genera and 14 species, and 24 isolates (89%) were identified as the genus Streptomyces. Many of the representative isolates had antimicrobial activities against plant pathogenic fungi and Gram-positive bacteria. In addition, most of the isolates (78%) showed biochemical properties to hydrolyze cellulose and casein. The results demonstrated that diverse and valuable actinomycetes could be isolated from insect fecal samples, indicating that insect guts can be rich sources for novel bioactive compounds.

• Journal of Life Science
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• v.14 no.3
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• pp.435-440
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• 2004

The gene encoding a extremely thermostable 4-$\alpha$-glucanotransferase from a hyperthermophilic archaeon, Thermococcus litoralis, was cloned, sequenced and expressed in Escherichia coli. The amino acid sequence of the enzyme was distantly related to other functionally-related ones, such as D-enzymes. The enzyme is of industrial interest because of a novel activity of producing cycloamylose and is also important for fundamental studies of protein, sugar-metabolizing enzymes. In this paper, the overexpression of 4-$\alpha$-glucanotransferase in E. coli was carried out expression vector system with lac and T7 promoters. The enzyme was successfully overexpressed, and purified by the heat treatment of a cell-free extract, successive Butyl-Toyopearl and Mono Q chromatographies. The purified recombinant enzyme showed the same specific activity and the same mobility in SDS-PAGE as natural enzyme.

• Journal of Life Science
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• v.18 no.2
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• pp.287-290
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• 2008

In order to obtain yeast cells producing a bacteriocin, Subpeptin JM4-A or Subpeptin JM4-B, the 48 bp oligonucleotides corresponding to Subpeptin JM4-A and Subpeptin JM4-B genes including codon for start and stop were chemically synthesized and cloned into pAUR123, an yeast expression vector. Transformed yeast cells exhibited growth inhibition of Bacillus subtilis, Escherichia coli and Pseudonomas aeruginosa. This result indicates that yeast cells producing Subpeptin JM4-A or Subpeptin JM4-B possess bacteriocidal properties against both Gram positive B. subtilis and Gram negative E. coli and P. aeruginosa cells. The recombinant yeast strains constructed in this study can be applied in the food preservative or. animal foodfeed.

• Biomedical Science Letters
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• v.4 no.1
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• pp.1-9
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• 1998

A Corynebacterium diphtheriae iron-repressible gene dirA, that was homologous to TSA of Saccharomyces cerevisiae and AhpC subunit of Salmonella typhimurium alkyl hydroperoxide reductase, was amplified with PCR and expressed in E. coli. The DirA purified from the transformed E. coli crude extracts prevented the inactivation of enzyme caused by metal-catalyzed oxidation (MCO) system containing thiols but not by ascorbate/Fe$^{3+}$/$O_2$ MCO system. The DirA concentration, which inhibited the inactivation of glutamine synthetase by 50% (IC$_{50}$) against MCO system, was 0.12 mg/ml. The multimeric forms of DirA were converted to the monomeric form in SDS-PAGE under the thioredoxin system comprised of NADPH, Saccharomyces cerevisiae thioredoxin reductase, and thioredoxin. Also, DirA showed thioredoxin dependent peroxidase activity. All of these results were consistent with the characteristics of a thiol specific antioxidant (TSA) protein having two conserved cysteine residues.

• The Korean Journal of Mycology
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• v.27 no.2 s.89
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• pp.132-137
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• 1999

This study was carried out to develop the molecular marker for the detection of Pseudomonas tolaasii, a causative agent of bacterial brown blotch disease of oyster mushroom (Pleurotus ostreatus). When several primers designed from repetitive sequences and pectin lyase genes of bacteria were used to produce DNA polymorphism from different Pseudomonas spp. isolated from edible mushrooms, PEU1 primer derived from pectin lyase gene produced polymorphic bands differentiating P. tolaasii strains from other Pseudomonas species. Two bands, 1.0kb and 0.4kb, found commonly in 6 isolates of P. tolaasii were cloned into pGEM-T vector which were designated as pPTOP1 and pPTOP2, respectively, to use as probe. The 0.4 kb insert of pPTOP2 hybridized to only 6 isolates of P. tolaasii, but did not to the other Pseudomonas species. As few as $1.5{\times}10^3$ colony forming unit (cfu) of P. tolaasii could be detected by dot blot hybridization with the cloned 0.4kb DNA in pPTOP2.

• Journal of Sericultural and Entomological Science
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• v.52 no.1
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• pp.25-32
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• 2014

We produced the transgenic silkworm that expressed the enhanced blue fluorescent protein (EBFP) in the cocoon of silkworms. The EBFP fusion protein, each with N- and C-terminal sequences of the fibroin H-chain, was designed to be secreted into the lumen of the posterior silk glands. The expression of the EBFP/H-chain fusion gene was regulated by the fibroin H-chain promoter. The use of the $3{\times}P3$-driven DsRed2 cDNA as a marker allowed us to rapidly distinguish transgenic silkworm. A mixture of the donor and helper vector was micro-injected into 300 eggs of silkworms, Baegokjam. We obtained 5 broods. The cocoon displayed blue fluorescence, proving that the fusion protein was present in the cocoon. Also, the presence of fusion proteins in cocoons was demonstrated by SDS-PAGE and western blot analysis. Accordingly, we suggest that the EBFP fluorescence silk will enable the production of the silk-based biomaterials.

• Korean Journal of Food Science and Technology
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• v.49 no.6
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• pp.610-616
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• 2017

${\gamma}$-Glutamyltranspeptidase (GGT, EC 2.3.2.2) transfers ${\gamma}$-glutamyl moiety from glutamine to amino acids or peptides and hydrolyzes glutamine to glutamate and ammonia. In order to overproduce ${\gamma}$-glutamyltranspeptidase from Bacillus amyloliquefaciens (BAGGT), the encoding gene was cloned and expressed in Bacillus subtilis. The productivity of BAGGT in Bacillus subtilis was improved by 42-fold by using a dual-promoter system that was generated by combining promoters from B. subtilis ${\alpha}$-amylase and BAGGT genes. Through optimization of medium composition by Plackett-Burman design and central composition design, BAGGT was produced at $18.3{\times}10^7U/L$ of culture in the optimized medium. Compared to previously used Luria-Bertani medium, the optimized culture medium (15 g/L molasses, 60 g/L corn steep liquor, 6 g/L yeast extract, 4 g/L NaCl, 6 g/L $K_2HPO_4$, and 2 g/L $KH_2PO_4$), resulted in a 4.3-fold increase in production of BAGGT.

• Microbiology and Biotechnology Letters
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• v.36 no.1
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• pp.49-54
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• 2008

The gene encoding human pancreatic pro-carboxypeptidase B (CPB) was cloned and fused to Saccharomyces cerevisiae mating factor alpha-1 secretion signal $(MF{\alpha}1)$, in which the transcription of $MF{\alpha}1$-pro-CPB was under the control of GAL10 promoter. The constructed plasmid $pY{\alpha}$-hproCPB(7.72 kb) was transformed into S. cerevisiae 2805. The recombinant human pro-CPB (hproCPB) was successfully expressed in S. cerevisiae after induction of galactose, and could be secreted into the culture medium. By analyses of SDS-PAGE and western blotting, the molecular weight of the purified hproCPB was estimated to be a 45.9kDa. The activity of extracellular hCPB after removal of pro-region by trypsin treatment reached about 10.16 unit/ml at batch culture of S. cerevisiae $2805/pY{\alpha}$-hproCPB for 60 h. Also, the Km value of partially purified recombinant hCPB is about 0.43 mM.