• 한국생물공학회:학술대회논문집
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• 2000.11a
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• pp.669-671
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• 2000

Antimicrobial cationic peptides have attracted increasing research and clinical interest as a natural antibiotics due to their broad spectrum of antimicrobial activites and the rapid development of multidrug-resistant pathogenic microorganisms. In this study, first, we synthesized artificial fusion partner and cationic peptide genes (lactoferricin, magainin, protegrin-1, and indolicidin). Second, we constructed recombinant expression vectors and then transformed Pichia pastoris. Finally, expressed cationic peptides were purified and tested for their antimicrobial activites. Antimicrobial activity has been tested upon the appearance of clearing zone on the plate with the lawn of gram negative E.coli XL- I blue and garm positive Staphylococcus aureus. Protegrin-1 and Indolicidin have apparant activity of cationic peotides. This fusion technique may lead to a general and suitable tool for production of pure antimicrobial cationic peptides in Pichia pastoris.

• Korean Journal of Pharmacognosy
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• v.31 no.2
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• pp.235-239
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• 2000

The effects of several compounds related to signal transduction cascade were determined to induce the production of alizarin and purpurin in the hairy root culture system of Rubia cordifolia var. pratensis. It was found that out of five tested compounds jasmonic acid(1 mg/l) and methyl jasmonate(1 mg/l) stimulated strongly the biosynthesis of the pigments while linolenic acid(1 mg/l) induced no significant increase of the product. Yeast extract(600 mg/l) and arachidonic acid(1 mg/l) showed relatively mild inducing effects on production of alizarin. The effects of jasmonic acid and methyl jasmonate were reduced by treatment with cycloheximide(2.8 mg/l).

• Biotechnology and Bioprocess Engineering:BBE
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• v.3 no.2
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• pp.55-60
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• 1998

Various Saccharomyces cerevisiae strains were transformed with a 2 ${\mu}$-based multicopy expression plasmid, pYIGP, carrying Kluyveromyces marxianus inulinase gene under the control of GAPDH promoter. Among then two strains, SEY2102 and 2805, showed high levels of cell growth and inulinase expression, and were selected to study their fermentation properties on inulin. Jerusalem artichoke inulin was more effective for cell growth (10∼11 g-dry wt./L at 48 hr) and inulinase expression (1.0 units/mL with SEY2102/pYIGP and 2.5 units/mL with 2805/pYIGP) than other inulin sources such as dahlia and chicory. It was also found that maximal ethanol production of 9 g/L was obtained from Jerusalem artichoke inulin at the early stationary phase (around 30 hr), indicating that recombinant S. cerevisiae cells secreting exoinulinase could be used for the simultaneous saccharification of inulin and ethanol fermentation.

• Journal of Life Science
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• v.22 no.2
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• pp.142-147
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• 2012

The sporulation-specific glucoamylase (SGA) of Saccharomyces diastaticus is known to be produced in the cytoplasm during sporulation. For the purpose of proving that SGA has secretory potential, we constructed a hybrid plasmid, pYESC25, containing the promoter and the putative signal sequence of the SGA fused in frame to the endo-1,4-${\beta}$-D-glucanase (CMCase) gene of Bacillus subtilis without its own signal sequence. The recipient yeast strain of S. diastaticus YIY345 was transformed with the hybrid plasmid. CMCase secretion from S. diastaticus harboring pYESC25 into culture medium was confirmed by the formation of yellowish halos around transformants after staining with Congo red on a CMC agar plate. The transformant culture was fractionated to the extracellular, periplasmic, and intracellular fraction, followed by the measurement of CMCase activity. About 63% and 13% enzyme activity were detected in the culture supernatant (extracellular fraction) and periplasmic fraction, respectively. Furthermore, ConA-Sepharose chromatography, native gel electrophoresis, and activity staining revealed that CMCase produced in yeast was glycosylated and its molecular weight was larger than that of the unglycosylated form from B. subtilis. Taking these findings together, SGA has the potential of secretion to culture medium, and the putative signal sequence of SGA can efficiently direct bacterial CMCase to the yeast secretion pathway.

• Korean journal of applied entomology
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• v.61 no.1
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• pp.101-108
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• 2022

Because of their specificity to target insects and relatively low toxicity to non-target organisms, insect growth regulators (IGRs) have been regarded as attractive alternatives to chemical insecticides. Commercially available IGRs are classified into juvenile hormone agonists (JHAs), ecdysone agonists (EAs), and chitin synthesis inhibitors (CSIs) according to their mode of action. Recently, JH-mediated interaction of methoprene-tolerant (Met), which is JH receptor, and its binding partners have been replicated in vitro using yeast cells transformed with the Met and FISC/CYC genes of A. aegypti. Using this in vitro yeast two-hybrid β-galactosidase assay, juvenile hormone antagonists (JHANs) have been identified from various sources including chemical libraries, plants, and microorganisms. As juvenile hormone (JH) is an insect specific hormone and regulates development, reproduction, diapause and other physiological processes, JHANs fatally disrupt the endocrine signals, which result in abnormal development and larval death. These results suggested that JHANs could be efficiently applied as IGR insecticides with a broad insecticidal spectrum. This review discuses JH signaling pathway mediated by Met and future prospects of JHANs as environmentally benign IGR insecticides.

• Journal of Life Science
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• v.15 no.4 s.71
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• pp.566-571
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• 2005

A natural habitat bacterium, Enterobacter cloaceae K41 was isolated from fresh water plant root and identified. This strain was used to investigate heavy metal resistance. The optimal growth conditions of the bacterium were LB medium containing$1\%$ yeast extract, $1\%$ lactose, $1\%$ NaCl, pH 7.0, at $37^{\circ}C$, and for 24 hours on a shaker. The minimal inhibitory concentration (MIC) of heavy metals against E. cloaceae KCTC2519 and E. cloaceae K41 was compared. The MIC of E. cloaceae K41 was 150 ppm in Cu, 50 ppm in Cd whereas that of the standard strain was 50 ppm in Cu but no growth was observed either Cd or two mixed heavy metal solution. The presence of plasmid was cleared from the isolated strain whereas no possession from the standard strain. The plasmid from E. cloaceae K41 was transformed into E. coli $DH5{\alpha}$. The MIC of transformed strain increased resistance 7 times in Cu and 6 times in Cd by insertion of this plasmid. The metal adsorption of the transformant was increased 1.3 times in Cu and 1.5 times in Cd indicating the plasmid was responsible for heavy metal resistance.

• Bulletin of the Korean Chemical Society
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• v.25 no.2
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• pp.237-242
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• 2004

Recombinant human ferritin homopolymers (rHF and rLF) were successfully produced in the Saccharomyces cerevisiae Y2805, which was transformed with human ferritin H or L-chain genes, respectively. In order to characterize the molecular properties of the recombinant ferritins in relation to mineralization, the proteins were isolated and apoferritins were prepared. The apoferritins were reconstituted with 2000 Fe atoms per protein molecule under various experimental conditions (the concentration of the protein, the buffer concentration of the MOPS buffer, the total volume of the reaction and the reconstitution method). The structure and composition of the iron cores formed in the ferritins were examined using transmission electron microscopy. The recombinant ferritins behaved in a similar manner to other mammalian ferritins in accumulating iron in the core. Proteins of rHF and rLF showed varying reconstitution yields of 37-72% depending on the reaction conditions. In general, the rHF showed higher reconstitution yield than the rLF at the protein concentrations and the reaction volumes we examined. Iron cores with a similar mean particle size were obtained in the rHF, rLF and horse spleen ferritin reconstituted at a protein concentration of 1.0 mg/mL. Electron diffraction of all the three ferritins showed 2-3 diffuse lines, with d-spacings corresponding to those of the mineral ferrihydrite with a limited crystallinity.

• Journal of Life Science
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• v.12 no.4
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• pp.496-503
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• 2002

To develop a yeast strain of Pichia pastoris producing an antibacterial peptide, we have attempted the expression and secretion of an insect defensin. The nucleotide sequences corresponding to mature defensin were chemically synthesized by 6 oligomers, assembled in vitro and the synthesized gene was identified by nucleotide sequencing. The prepro sequence of yeast mating factor $\alpha$1 and the defensin gene were recombined into a Pichia expression vector, pPIC9K. The resulting plasmid, pPIDE, was transformed into P. pastoris GSl15 and transformants selected on histidine-deficient minimal plates were tested for antibacterial activity against Micrococcus luteus. Four strains with different antibacterial activity were selected for further analysis. Southern hybridization and RT-PCR verified the defensin gene was maintained and transcribed in a host. Four strains were cultivated in YPD broth for 96 hours to compare cell growth and antibacterial activity, They showed no difference in cell growth, however, each strain showed different antibacterial activity pattern with culture time. The maximal activity was about 550 AU/ $m\ell$.

• KSBB Journal
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• v.11 no.3
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• pp.374-379
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• 1996

The production of extracellular mannitot by an efficient mannitol-producing bacterium, Lactobacillus sp. KY-107 was studied in shake flask culture using the modified MRS medium. Maximum mannitol production was obtained with fructose as the sole carbon source. Within 95 hours of incubation, a final concentration of 70g/L of mannitol from 100g/L fructose was obtained with an indicated yield of 86% based on fructose consumed. However, higher concentrations of fructose could not effectively be transformed to mannitol due to a lack of osmotolerance. The strain produced no other polyols such as glycerol and sorbitol as by-products. Yeast extract was best nitrogen source and high levels of inorganic phosphate up to 10g/L did not show any detrimental effect for mannitol formation. Manganese ion played important role in both cell growth and mannitol production. The optimum culture temperature and initial pH were $35^{\circ}C$ and 6-8, respectively.

• Journal of Microbiology and Biotechnology
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• v.25 no.11
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• pp.1856-1862
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• 2015

In order to improve the stability of endoglucanase under thermal and acidic conditions, the endoglucanase gene was fused to the N-terminus of the Saccharomyces cerevisiae pir gene, encoding the cell wall protein PIR. The fusion gene was transformed into Pichia pastoris GS115 for expression. A resulting strain with high expression and high activity was identified by examining resistance to Geneticin 418, Congo red staining, and quantitative analysis of enzyme activity. SDS-PAGE analysis revealed that the endoglucanase was successfully displayed on the yeast cell surface. The displayed endoglucanase (DEG) showed maximum activity towards sodium carboxyl methyl cellulose at approximately 275 IU/g cell dry weight. DEG exhibited greater than 60% residual activity in the pH range 2.5-8.5, higher than free endoglucanase (FEG), which had 40% residual activity at the same pH range. The highest tolerated temperature for DEG was 70℃, much higher than that of FEG, which was approximately 50℃. Moreover, DEG showed 91.1% activity at 65℃ for 120 min, while FEG only kept 77.8% residual activity over the same period. The half-life of DEG was 270 min at 65℃, compared with only 150 min for FEG. DEG could be used repeatedly at least three times. These results suggest that the DEG has broad applications as a yeast whole-cell biocatalyst, due to its novel properties of high catalytic efficiency, acid-thermal stabilities, and reusability.