• KSBB Journal
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• v.30 no.3
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• pp.103-108
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• 2015

Production of foreign proteins by transgenic plant cell cultures has several advantages such as post-translational modification, low risk of product contamination and low-cost production and purification. However, target proteins are degraded by extracellular proteases existing in the media. A solution to this problem is the use of perfusion culture and ion exchange chromatography for the application of integrated bioprocess using in situ recovery. With this method, production of human granulocyte-macrophage colony-stimulating factor (hGM-CSF) was investigated in this study. First, optimization of cell concentration during the induction phase for the production of hGM-CSF was examined. As cell concentration increased, the level of hGM-CSF was decreased due to the presence of extracellular proteases. Induction using sugarfree media produced 33% more hGM-CSF. The effects of pH on the binding of hGM-CSF to cationic and anionic exchange resins were also investigated. In terms of stability, optimal pH was found to be 5~7. In the case of using buffer exchange when CM-Sepharose was used as a cationic exchange resin, optimal pH for binding was 4.8 and adsorption yield was 77%. When DEAE-Sepharose was used as an anionic exchange resin, it was 5.5 (74%). Without buffer exchange, optimal pH was 4.6 (84%). From these results, an integrated bioprocess using in situ recovery with simultaneous production and separation of foreign protein in transgenic plant cell suspension cultures was found to be feasible.

• Korean Journal of Microbiology
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• v.48 no.1
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• pp.48-51
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• 2012

A novel Chryseobacterium sp. JK1 strain producing extracellular protease had been isolated from soil. The largest clear zones were observed on nutrient agar plates supplemented with 1% skim milk at $30-35^{\circ}C$ along with the growth of Chryseobacterium sp. JK1. The cell growth of JK1 strain was maximal at 24 h and maximum protease activity was reached up to 560 unit/ml at the stationary phase in liquid culture. In the presence of maltose, glucose or mannitol in Nutrient broth, cells grew well, but protease were produced poorly with lower production yields of 64-77% than in NB broth only. Similarly, the addition of skim milk, beef extract, yeast extract, malt extract or tryptone showed good growth and poor enzyme production. On the contrary, the addition of $(NH_4)_2HPO_4$ or $(NH_4)_2SO_4$ gave poor growth and good enzyme production of 121-146%.

• Mycobiology
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• v.33 no.1
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• pp.23-29
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• 2005

The effects of different concentrations of three amino acids as carbon and or nitrogen sources on mycelial dry weights, changes in pH values of synthetic medium, ammonia secretion and extracellular protease activity by three zoosporic fungi, pathogens of fish and shellfish, were studied. As compared with the control, the addition of isoleucine and aspartic acid as nitrogen sources were generally stimulative for mycelial dry weight production whereas phenylalanine was inhibitory irrespective to the tested fungal species. When amino acids served as carbon and nitrogen sources, the mycelial dry weights of the three fungi were increased (mostly non-significantly) relative to untreated control but weights were decreased as the concentrations of the three amino acids raised. The addition of individual amino acids as carbon and nitrogen sources to the medium significantly increased pH values of the medium comparable to the control. The addition of each of the three amino acids as carbon and nitrogen sources to the medium significantly induced ammonia secretion by the three species of zoosporic fungi. Ammonia secretion in synthetic medium amended with amino acids as nitrogen source raised by the three zoosporic fungi relative to untreated control except in case of Achlya racemosa treated with isoleucine. Extracellular protease activity was almost promoted in case of Achlya proliferoides and Saprolegnia furcata cultures treated with isoleucine and aspartic acid individually in presence of glucose and vice versa in case of phenylalanine. However, extracellular protease activity of A. racemosa decreased compared with the control at various concentrations of isoleucine and both phenylalanine and aspartic acid assumed inconsistent effects. Extracellular protease activity of the three zoosporic fungi in the medium devoid of glucose varied depending upon zoosporic fungal species, the tested amino acid and the applied concentrations. The values of protease activity were approximately less two folds than that obtained in presence of glucose.

• Molecules and Cells
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• v.39 no.3
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• pp.242-249
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• 2016

A balance between production and degradation of reactive oxygen species (ROS) is critical for maintaining cellular homeostasis. Increased levels of ROS during oxidative stress are associated with disease conditions. Antioxidant enzymes, such as extracellular superoxide dismutase (EC-SOD), in the extracellular matrix (ECM) neutralize the toxicity of superoxide. Recent studies have emphasized the importance of EC-SOD in protecting the brain, lungs, and other tissues from oxidative stress. Therefore, EC-SOD would be an excellent therapeutic drug for treatment of diseases caused by oxidative stress. We cloned both the full length (residues 1-240) and truncated (residues 19-240) forms of human EC-SOD (hEC-SOD) into the donor plasmid pFastBacHTb. After transposition, the bacmid was transfected into the Sf9-baculovirus expression system and the expressed hEC-SOD purified using FLAG-tag. Western blot analysis revealed that hEC-SOD is present both as a monomer (33 kDa) and a dimer (66 kDa), as detected by the FLAG antibody. A water-soluble tetrazolium (WST-1) assay showed that both full length and truncated hEC-SOD proteins were enzymatically active. We showed that a potent superoxide dismutase inhibitor, diethyldithiocarbamate (DDC), inhibits hEC-SOD activity.

• 한국생물공학회:학술대회논문집
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• 2001.11a
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• pp.270-273
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• 2001

The fluorescence sensor is utilized to monitor the complex fluorescence patterns of intra- and extracellular components in cultivation processes. Especially biogenic fluorophores such as proteins and peptides (tryptophan, phenylalanine), coenzymes (FAD, NAD(P)H) and vitamins (riboflavin, pyridoxine) within cells are detected by a fluorescence sensor. In this work a 2-dimensional fluorescence sensor has been used to monitor a production process of itaconic acid by Aspergillus terreus and the on-line monitored spectra data can be con-elated to off-line data measured by a few methods.

• Proceedings of the PSK Conference
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• 2002.10a
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• pp.331.1-331.1
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• 2002

Actinomycetes CS0703 has been isolated in soil sample from location in the Jeju province. Korea. and produces alkaline extracellular proteases. To maximize protease production, initial pH of the culture medium was adjusted to 12.0 with NaOH and incubated at $48^{\circ}C$ on a rotary shaking incubator(180rpm). Actinomycetes CS0703 produced high level of protease at late exponential phase when grown in OSYM medium (oatmeal 2.0%. soybean meal 1%. dried yeast 1%. mannitol 1%). (omitted)

• Food Science and Preservation
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• v.17 no.3
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• pp.410-417
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• 2010

Several extracellular protease-producing bacteria were isolated from Chungkookjang, a traditional Korean food of fermented soybeans, on skim milk agar plates. Among these bacteria, strain D14 exhibited the highest production (15.2 U/mL) and specific activity (40.0 U/mg protein) of extracellular protease activity as assessed on growth in a protease induction medium composed of 1% (w/v) soluble starch, 1.5% (w/v) skim milk, 0.5% (w/v) yeast extract, and 2% (w/v) NaCl. The bacterium was identified as Bacillus subtilis based on morphological and physiological characteristics and 16S rDNA sequence. A BLAST search of 16S rDNA sequences revealed that the isolate was most closely related to Bacillus subtilis subsp. subtilis strain NCIB 3610. The 16S rDNA sequence homology was 99.9%. Our isolate produced the highest level of protease when grown in a protease induction medium containing 1% (w/v) sorbitol and 0.5% (w/v) yeast extract. Fructose and glucose reduced enzyme production to 12.7% and 35.9%, respectively, of the level seen when the strain was grown in medium containing soluble starch. Soytone also reduced enzyme production to 61.4% of the level noted when the strain was grown in medium containing yeast extract.

• Korean Journal of Ecology and Environment
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• v.40 no.2
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• pp.294-302
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• 2007

This study was to evaluate microcystin production by Microcystis aeruginosa in response to three different levels of indirect (0, 10, 50% of fish cultured media filtrate; control, FCMF1 and FCMF2) exposures to omnivorous and planktivorous fish (Carassius gibelio langsdorfi and Hypophthalmichthys molitrix, CCMF and HCMF, repectively). The cell biomass, intracellular microcystin (MC) and extracellular MC were measured everyday. The intracellular MC contents of all treatments were significantly increased than the controls (CCMF1, P=0.015; CCMF2, P<0.001; HCMF1, P<0.001; HCMF2, P<0.001). The intracellular MC contents of M. aeruginosa were significantly higher in CCMF2 than in CCMF1 (P=(0.023), Those of M, aeruginosa in HCMF2 were significantly higher than that in HCMF1 (P<0.001). The extracellular MC contents were not significantly different between control and CCMFs but those of M, aeruginosa in HCMF1 and HCMF2 were significantly higher than that in control (HCMF1, P=0.003; HCMF2, P<0.001). This study strongly supports that induced-defensive MC production (intra and extracellular MC) of potentially toxic cyanobacteria in response to kairomone concentration and this results can consider the biomanipulation of eutrophic waters as well as an information concerning strategies for recovering eutrophic waters.

• Microbiology and Biotechnology Letters
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• v.47 no.4
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• pp.603-613
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• 2019

This study was carried out to examine the antagonistic effect against phytopathogenic fungi of isolated strains from soil samples collected from Busan, Changwon, and Jeju Island: Botrytis cinerea, Colletotrichum acutatum, Corynespora cassiicola, Fusarium sp., Rhizoctonia solani, Phytophthora capsici, and Sclerotinia sclerotiorum. According to results of our studies, isolated strains showed an antagonistic effect against phytopathogenic fungi. Such an antagonistic effect against phytopathogenic fungi is seen due to the production of siderophores, antibiotic substances, and extracellular amylase, cellulase, protease, and xylanase enzyme activities. Extracellular enzymes produced by isolated strains were significant, given that they inhibited the growth of phytopathogenic fungi by causing bacteriolysis of the cell wall of plant pathogenic fungi. This is essential to break down the cell wall of plant pathogenic fungi and thus help plant growth by converting macromolecules, which cannot be used by the plant for growth, into small molecules. In addition, they are putative candidates as biological agents to promote plant growth and inhibit growth of phytopathogenic fungi through nitrogen fixation, indole-3-acetic acid production, siderophore production, and extracellular enzyme activity. Therefore, this study suggests the possibility of using Bacillus subtilis ANGa5, Bacillus aerius ANGa25, and Bacillus methylotrophicus ANGa27 as new biological agents, and it is considered that further studies are necessary to prove their effect as novel biological agents by standardization of formulation and optimization of selected effective microorganisms, determination of their preservation period, and crop cultivation tests.

• Asian-Australasian Journal of Animal Sciences
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• v.2 no.3
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• pp.444-445
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• 1989