• Title/Summary/Keyword: immobilized cell

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Organic Solvent Stable Lipase from Pseudomonas sp. BCNU 171 (Pseudomonas sp. BCNU 171이 생산하는 유기용매 내성 리파아제)

  • Choi, Hye Jung;Kwon, Gi-Seok;Joo, Woo Hong
    • Journal of Life Science
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    • v.25 no.3
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    • pp.345-348
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    • 2015
  • An organic solvent stable lipase from solvent-tolerant Pseudomonas sp. BCNU 171 had an optimal pH of 8 and an optimal temperature of 37℃. This crude extracellular lipase from BCNU 171 exhibited increased stability in the presence of various types of solvents at high concentrations (25%, v/v). The lipase stability was found to be highest in the presence of xylene (137%), followed by toluene (131%), octane (130%), and butanol (104%). Overall, BCNU 171 lipase tended to be more stable than immobilized commercial lipase (Novozyme435) in the presence of organic solvents. Furthermore, BCNU 171 lipase maintained about 90% of its enzyme original activity in the presence of NH4+, Na+, Ba2+, Hg2+, Ni2+, Cu2+, and Ca2+ion and significantly increased its enzyme activity in the presence of various emulsifying agents. Thus, the organic solvent stable lipase from Pseudomonas sp. BCNU 171 could be usable as a potential whole cell biocatalyst and for synthetic applications of enzymes for industrial chemical processes in organic solvents without using immobilization.

Improvement of L-Lysine Productivity by Using Cell Fusion and Immobilized System (세포융합과 고정화 시스템을 이용한 L-Lysine의 생산성 향상)

  • Ryu, Beung-Ho;Kim, Hye-Sung;Roh, Myung-Hoon;Park, Bob-Gyu;Chung, Jong-Soon;Bai, Ki-Chul
    • Korean Journal of Food Science and Technology
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    • v.21 no.1
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    • pp.154-163
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    • 1989
  • This studies were designed to improve the productivity of L-lysine by protoplast fusion and immobilized system of fusants using strains of Brevibacterium flavum ATCC 21528, Brevibacterium lactofermentum ATCC 21086 and Corynebacterium glutamicum 820. Mutants were isolated with concentration method of $300{\mu}g/ml$ penicillin-G after treatment of $250{\mu}g/ml$ N-methyl-N-nitro-N-nitrosoguanidine. B. flavum $37-2(Hos^-,\;Kan^r,\;AEC^r)$, B. lactofermentum $6-2(Ile^-,\;Val^-,\;Str^r,\;AEC^r)$ and C. glutamicum 57-5$(Met^-,\;Thr^-,\;Rif^r,\;AEC^r)$ were isolated from mutants. Protoplasts were induced by being incubated with $500{\mu}g/ml$ lysozyme of lysis solution for 6 hr and the ratio of protoplast formation and regeneration were ranging from 97-99% and 33-37%, respectively. Fusion frequencies of fusants of BBFL 21, BCFG 37 and BCLG 59 were shown in the range from $1.25{\times}10^{-6}\;to\;5.83{\times}10^{-7}$ under the optimum conditions. The fusant BBFL 21 showed the highest productivity of $411.1\;ng/ml{\cdot}hr$ L-lysine in the lysine productivity broth at $30^{\circ}C$ for 72hr. In the immobilization systems, fusant BBFL 21 was employed in various polymer matrices such as sodium alginate, polyacrylamide, agar and ${\alpha}-carrageena$. The immobilization of sodium alginate showed the highest productivity of $413\;ng/ml{\cdot}hr$ L-lysine in the batch system. Continuous fermentation of immobilization system by using tube fermentor was produced the highest productivity $416.7\;ng/ml{\cdot}hr $ L-lysine under optimum condition.

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Evaluating the Removal Efficiency of Organic Compounds and Nitrogen Depending on Loading Rate in Wastewater Treatment from Fisheries Processing Plant Using an Entrapped Mixed Microbial Cell Technique (미생물 강제포획기술을 이용한 수산물 가공공장 폐수처리에서 부하율에 따른 유기물 및 질소의 제거 효율성 평가)

  • Jeong Byung-Cheol;Chang Soo-Hyun;Jeong Byung-Gon
    • Journal of the Korean Society for Marine Environment & Energy
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    • v.9 no.1
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    • pp.14-20
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    • 2006
  • In this study, the feasibility of simultaneous removal of organic materials and nitrogen in the waste-water from fisheries processing plant was evaluated using entrapped mixed microbial cell technique(EMMC) process. The experiment was performed using activated sludge from municipal sewage treatment plant which was immobilized with gel matrix by cellulose triacetate. It was found that the stable operation at the treatment system which is composed of anoxic and oxic tank, was possible when the organic and nitrogen loading rates were increased stepwise. The organic and nitrogen loading rates were applied from 0.65 to $1.72kgCOD/m^3/d$ and from 0.119 to $0.317kgT-N/m^3$ with four steps, respectively. The maximum nitrogen loading rate which could satisfy the regulated effluent standard of nitrogen concentration, was $0.3kgT-N/m^3/d$. The removal efficiency of total nitrogen was decreased apparently as increasing nitrogen loading rates, whereas the removal efficiency of ammonium nitrogen was effective at the all tested nitrogen loading rates. Therefore, it was concluded that nitrification was efficient at the system. Nitrate removal efficiency ranged from 98.62% to 99.51%, whereas the nitrification efficiency at the oxic tank ranged 94.0% to 96.9% at the tested loading rates. The removal efficiencies of chemical oxygen demand(COD) and those of total nitrogen at the entire system ranged from 94.2% to 96.6% and 73.4% to 83.4%, respectively.

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Soluble Prokaryotic Expression and Purification of Bioactive Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand

  • Do, Bich Hang;Nguyen, Minh Tan;Song, Jung-A;Park, Sangsu;Yoo, Jiwon;Jang, Jaepyeong;Lee, Sunju;So, Seoungjun;Yoon, Yejin;Kim, Inki;Lee, Kyungjin;Jang, Yeon Jin;Choe, Han
    • Journal of Microbiology and Biotechnology
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    • v.27 no.12
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    • pp.2156-2164
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    • 2017
  • Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is considered as an antitumor agent owing to its ability to induce apoptosis of cancer cells without imparting toxicity toward most normal cells. TRAIL is produced in poor yield because of its insoluble expression in the cytoplasm of E. coli. In this study, we achieved soluble expression of TRAIL by fusing maltose-binding protein (MBP), b'a' domain of protein disulfide isomerase (PDIb'a'), or protein disulfide isomerase at the N-terminus of TRAIL. The TRAIL was purified using subsequent immobilized metal affinity chromatography and amylose-binding chromatography, with the tag removal using tobacco etch virus protease. Approximately 4.5 mg of pure TRAIL was produced from 125 ml flask culture with a purification yield of 71.6%. The endotoxin level of the final product was $0.4EU/{\mu}g$, as measured by the Limulus amebocyte lysate endotoxin assay. The purified TRAIL was validated and shown to cause apoptosis of HeLa cells with an $EC_{50}$ and Hill coefficient of $0.6{{\pm}}0.03nM$ and $2.41{\pm}0.15$, respectively. The high level of apoptosis in HeLa cells following administration of purified TRAIL indicates the significance and novelty of this method for producing high-grade and high-yield TRAIL.

Production, Immobilization, and Characterization of Croceibacter atlanticus Lipase Isolated from the Antarctic Ross Sea (남극 로스해에서 분리한 Croceibacter atlanticus균 유래 리파아제의 생산, 고정화, 효소특성 연구)

  • Park, Chae Gyeong;Kim, Hyung Kwoun
    • Microbiology and Biotechnology Letters
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    • v.46 no.3
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    • pp.234-243
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    • 2018
  • The Antarctic Ocean contains numerous microorganisms that produce novel biocatalysts that can have applications in various industries. We screened various psychrophilic bacterial strains isolated from the Ross Sea and found that a Croceibacter atlanticus strain (Stock No. 40-F12) showed high lipolytic activity on a tributyrin plate. We isolated the corresponding lipase gene (lipCA) by shotgun cloning and expressed the LipCA enzyme in Escherichia coli cells. Homology modeling of LipCA was carried out using the Spain Arreo lake metagenome alpha/beta hydrolase as a template. According to the model, LipCA has an ${\alpha}/{\beta}$ hydrolase fold, Gly-X-Ser-X-Glymotif, and lid sequence, indicating that LipCA is a typical lipase enzyme. Active LipCA enzyme was purified fromthe cell-free extract by ammonium sulfate precipitation and gel filtration chromatography. We determined its enzymatic properties including optimum temperature and pH, stability, substrate specificity, and organic solvent stability. LipCA was immobilized by the cross-linked enzyme aggregate (CLEA) method and its enzymatic properties were compared to those of free LipCA. After cross-linking, temperature, pH, and organic solvent stability increased considerably, whereas substrate specificities did not changed. The LipCA CLEA was recovered by centrifugation and showed approximately 40% activity after 4th recovery. This is the first report of the expression, characterization, and immobilization of a C. atlanticus lipase, and this lipase could have potential industrial application.

The Effect of Oxygen Supply on the Production of Citric Acid from Encapsulated Aspergillus niger (산소공급이 캡슐고정화 Aspergillus niger의 구연산 생산에 미치는 영향)

  • Park, Joong-Kon;Jeong, Geung-Sik
    • KSBB Journal
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    • v.14 no.6
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    • pp.672-676
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    • 1999
  • Encapsulated Aspergillus niger was prepared in order to inspect the effect of oxygen supply on the production of citric acid. A. niger cells which had been immobilized in the calcium alginate capsule grew and mycellia penetrated through the capsule membrane after two days of cultivation and covered over all of the capsule after eight days. The mycellia became loose when the nitrogen source was sufficient of oxygen was deficient. The larger amount of encapsulated cells were put into a given growth medium, the smaller quantity of citric acid was produced. The increase of volumetric oxygen transfer coefficient from 1.8 $hr^-$ to 2.55 $hr^-$ in the flask culture accelerated cell growth rate but did not influence the production of citric acid. The high oxygen supply rate($k_La:\;150\;hr^-$) in the concentric air lift reactor hastened the growth of cells and hindered the production of the citric acid. The reduction of nitrogen source level in the growth medium in the concentric air lift reactor increased citric acid production by 40 percent of that of flask cultivation and the culture period was shortened by 3 days. The variation of the geometry of the concentric air lift reactor did not influence the growth rate of encapsulated cells and production rate of citric acid.

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Effects of the immobilization of heparin and rhPDGF-BB to titanium surfaces for the enhancement of osteoblastic functions and anti-inflammation

  • Huh, Jung-Bo;Lee, Jeong-Yo;Lee, Kyung-Lae;Kim, Sung-Eun;Yun, Mi-Jung;Shim, Ji-Suk;Shim, June-Sung;Shin, Sang-Wan
    • The Journal of Advanced Prosthodontics
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    • v.3 no.3
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    • pp.152-160
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    • 2011
  • PURPOSE. This study was to investigate the effects of recombinant human platelet-derived growth factor (rhPDGF-BB) and heparin to titanium surfaces for enhancement of osteoblastic functions and inhibition of inflammation activity. MATERIALS AND METHODS. The anodized titanium discs, not coated with any material, were used as a control group. In heparinized-Ti group, dopamine was anchored to the surface of Ti substrates, and coated with heparin. In PDGF-Ti group, rhPDGF-BB was immobilized onto heparinized Ti surface. The surface morphologies were investigated by the scanning electron microscope in each group. The release kinetics of rhPDGF-BB were analyzed, and cytotoxicity tests for each group were conducted. The biocompatibilities were characterized by measuring cell proliferation, alkaline phosphatase activity, and calcium deposition using MG-63 cells. Statistical comparisons were carried out by one-way ANOVA tests. Differences were considered statistically significant at $^*$P<.05 and $^{**}$P<.001. RESULTS. The combination of rhPDGF-BB and heparin stimulated alkaline phosphatase activity and OCN mRNA expression in osteoblastic cells ($^*$P<.05 and $^{**}$P<.001). MG-63 cells grown on PDGF-Ti had significantly higher amounts of calcium deposition than those grown on anodized Ti ($^{**}$ P<.001). Heparinized Ti was more anti-inflammatory compared to anodized Ti, when exposed to lipopolysaccharide using the transcript levels of TNF-${\alpha}$ and IL-6 of proinflammatory cytokine ($^*$P<.05 and $^{**}$P<.001). CONCLUSION. The result of this study demonstrated that the incorporation of rhPDGF-BB and heparin onto Ti surface enhanced osteoblastic functions and inhibited inflammation.

Comparative Analysis of Muscle Proteome from Porcine White and Red Muscles by Two-dimensional Electrophoresis (이차원전기영동법을 이용한 white muscle과 red muscle간의 단백질 발현양상의 비교분석)

  • Kim, N.K.;Joh, J.H.;Chu, K.S.;Park, H.R.;Park, B.Y.;Kim, O.H.;Lee, C.S.
    • Journal of Animal Science and Technology
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    • v.45 no.5
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    • pp.731-738
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    • 2003
  • The technique known as proteomics is useful for characterizing the protein expression pattern of a particular tissue or cell type as well as quantitatively identifying differences in the levels of individual proteins. In present study, we carried out the comparative expression patterns of white and red muscles. We used the two-dimensional electrophoresis(2-DE) for analyzing the protein expression. Proteins isolated from porcine white and red muscles were separated by 12% poly-acrylamide gel and then were detected by coomassie blue and silver staining. More than 600 protein spots were detected on each 2-DE gel. By visual analysis of the stained gel, five proteins were identified to be differentially expressed in the white vs red muscle. By database searching based on the molecular weights and pI(isoelectric point) of the five proteins, three of them were found to be most close to troponin I, T and myoglobin. However, further researche is needed for identification and functional analysis of the unidentified proteins. In conclusion, we found five proteins, which are differentially expressed in the white vs red muscle. The functional analysis of the differentially expressed proteins will provide valuable information on biochemical characteristics of the muscle type.

Expression of the lux Genes in Escherichia coli for the Basis of Development of Biosensor (바이오센서 개발을 위한 발광 유전자의 대장균에서의 발현 시스템 분석)

  • Cho, Mi-Mi;Kim, Young-Doo;Kang, Kyung-Sook;Kim, Sook-Kyung;Yang, In-Chul;Park, Sang-Ryoul;Lee, Chan-Yong
    • Korean Journal of Microbiology
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    • v.45 no.4
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    • pp.419-424
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    • 2009
  • To provide the basis of biosensor based on the lux genes from bioluminescent bacteria of Photobacterium leiognathi and Vibrio harveyi, we test the expression of lux genes in several strains of Escherichia coli. The expression of the recombinant plasmid of PlXba.pT7-3, containing all lux genes requiring for light emission without adding substrate, in E. coli 43R was so strong to see the blue-green light in single colony as well as in the alginate immobilized cell. In addition, the light intensity was decreased by adding heavy metal ion such as cadmium and zinc ions. These result raise the possibility that a biosensor can be developed using the lux genes system.

Detection of Campylobacter jejuni in food and poultry visors using immunomagnetic separation and microtitre hybridization

  • Simard, Ronald-E.
    • Proceedings of the Korean Society of Fisheries Technology Conference
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    • 2000.05a
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    • pp.71-73
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    • 2000
  • Campylobacter jejuni is most frequently identified cause of cause of acute diarrhoeal infections in developeed countries, exceeding rates of illness caused by both salmonella and shigilla(Skirrow, 1990 ; Lior 1994). Previous studies on campylobacter jejuni contamination of commercial broiler carcasses in u.s.(Stern, 1992). Most cases of the disease result from indirect transmission of Campylobactor from animals via milk, water and meat. In addition to Campylobactor jejuni. the closely relates species Campylobactor coli and Campylobactor lari have also been implicated as agents of gastroenteritis in humans. Campylobactor coli represented only approximately 3% of the Campylobactor isolates from patients with Campylobactor enteritis(Griffiths and Park, 1990) whereas Campylobactor coli is mainly isolated from pork(Lmmerding et al., 1988). Campylobactor jejuni has also been isolated from cases of bacteremia, appendicitis and, recently, has been associated with Guillai-Barre syndrome(Allos and Blaser, 1994; von Wulffen et al., 1994; Phillips, 1995). Studies in volunteers indicated that the infectious dose for Campylobactor jejuni is low(about 500 organisms)(Robinson, 1981). The methods traditionally used to detect Campylobactor ssp. in food require at least two days of incubation in an enrichment broth followed by plating and two days of incubation on complex culture media containing many antibiotics(Goossens and Butzler, 1992). Finnaly, several biochemical tests must be done to confirm the indentification at the species level. Therfore, sensitive and specific methods for the detection of small numbers of Campylobactor cells in food are needed. Polymerase chain reaction(PCR) assays targeting specific DNA sequences have been developed for the detection of Campylobactor(Giesendorf and Quint, 1995; Hemandex et al., 1995; Winter and Slavidk, 1995). In most cases, a short enrichment step is needed to enhance the sensitivity of the assay prior to detection by PCR as the number of bacteria in the food products is low in comparison with those found in dinical samples, and because the complex composition of food matrices can hinder the PCR and lower its sensitivity. However, these PCR systems are technically demanding to carry out and cumbersome when processing a large number of samples simutaneously. In this paper, an immunomagnetic method to concentrate Campylobactor cells present in food or clinical samples after an enrichment step is described. To detect specifically the thermophilic Campylobactor. a monoclonal antibody was adsorbed on the surface of the magnetic beads which react against a major porin of 45kDa present on the surface of the cells(Huyer et al., 1986). After this partial purification and concentration step, detection of bound cells was achieved using a simple, inexpensive microtitre plate-based hybridization system. We examined two alternative detection systems, one specific for thermophilic Campylobactor based on the detection of 23S rRNA using an immobilized DNA probe. The second system is less specific but more sensitive because of the high copy number of the rRNA present in bacterial cell($10^3-10^4$). By using specific immunomagnetic beads against thermophilic Campylobactor, it was possible to concentrate these cells from a heterogeneous media and obtain highly specific hybridization reactions with good sensitivity. There are several advantages in using microtitre plates instead of filter membranes or other matrices for hybridization techniques. Microtitre plates are much easier to handle than filter membranes during the adsorption, washing, hybridization and detection steps, and their use faciilitates the simultanuous analysis of multiple sample. Here we report on the use of a very simple detection procedure based on a monoclonal anti-RNA-DNA hybrid antibody(Fliss et al., 1999) for detection of the RNA-DNA hybrids formed in the wells.

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