Earticle

Twelve bacterial strains capable of growing on phenol minimal medium were isolated from iron foundry activated sludge by enrichment culture, and amount them, one isolate which was the best in cell growth and phenol degradation was selected and identified as Acinetobacter junii POH. The optimal temperature, initial pH and phenol concentration in the above medium were 30℃, 7.5 and 1000 ppm, respectively. Cell growth of Acinetobacter junii POH dramatically increased 20 hrs cultivation-time and reached a almost stationary phsae 40 hrs cultivation-time then phenol was degraded about 98%. Cell growth was inhibited y phenol at concentrations over 1500 ppm. The isolate was resistant to several antibiotics as well as various heavy metal ions. The growth-limiting log P value of Acinetobacter junii POH on organic solvents was 2.9 in the LB medium. Therefore, it is suggested that Acinetobacter junii POH could be effectively used for the biological treatment of wastewater containing the presence of heavy metal ions and organic solvents.

Protein affinity membrane was prepared via the coating of chitosan gel on the porous flat polysulfone membrane surface, followed by the immobilization f the reactive dye (Cibacron Blue 3GA) to the chitonsan gel. The maximum protein binding capacity of affinity membrane was about 70ug/cm2 determined by the batch adsorption experiments of human serum albumin (HSA). Using module of this membrane, the characteristics of protein chromatography were investigated through the experiments of elution and frontal chromatography of HSA. This membrane module promises as a chromatography column, since it represented a lower pressure drop and a greater reproducibility. The protein separation ratio was significantly influenced by the flow rate of mobile phase and the injection quantity of HSA. The dynamic protein binding capacity of module decreased from the equilibrium binding capacity with increasing flow rate and approached the value of 15 - 20 ug/cm2 for flow rates above 6 mL/min.

Using rlFN- and rhGH as the model proteins, the refolding performances of the published processes were evaluated and compared. Key engineering parameters such as the type of denaturant and this concentration, protein concentration in the refolding buffer, and pH and ionic strength of the buffer were experimentally investigated. Furthermore, the role of a co-solvent of surfactant type in aggregation reduction was also studied. Of the denaturants tested (8M urea, 6M guanidine HCI, 0.5% SDS), SDS at alkaline pH (9.5) and ambient temperature gave the highest recovery yield. The SDS process was effective in the refolding of observed where dissolution proceeded better under lower strength (10 mM) but aggregation was suppressed under higher strength (>50 mM.) When PEG-4000 and/or Tween were added as co-solvent or refolding-enhancing additive, 1.6-2 times higher yield was realized. The‘masking’of the hyrophobic patches located on the surface of the protein with the surfactant molecules was believed to be responsible for the considerable reduction in aggregation during refolding.

To remove the low concentration of methane biologically, a three phase fluidized bed bioreactor immobilized with Methylosinus trichosporium OB3b was used. Optimum pH, temperature and bed height for the operation were pH7.0, 30℃ and 150cm, respectively. For the inlet methane concentration of 100-400ppm and flow rate of 2-4L/min, the removal efficiencies of the bioreactor using the activated carbon as a carrier were the range of 54-71%, whereas those using the biosand were the range of 45-56%. It was found that activated carbon was more efficient than the biosand for the removal of methane. When aeration tank was equipped with the bioreactor, the removal efficiency increased to 6-13% and maximum removal rate obtained in the experiment was 1184mg.CH4/min.

키토산 올리고당을 효율적으로 생산하기 위하여 고정화 효소 를 이용한 키토산의 효소적 가수분해를 시도하였다. Chitosanase는 Chitopearl계 고정화 담체에 대해서 높은 흡착율로 결합되었다. 키틴에 고정화된 효소는 비록 흡착율은 낮았지 만 그 활성은 가장 높게 나타났다. 키틴 고정화 효소는 유리 효소에 비해 약 90% 이상의 활성을 유지하였다. 고정화 효소의 최적 온도는 60°C로서 유리 효소보다 15℃ 더 높았으며, 열에 대한 안정성도 유리 효소보다 넓은 온도범위에서 우수하였다 그러나 고정화 효소는 pH에 대해서는 어떠한 뚜렷한 효과도 보이지 않았다. 고정화 효소의 저장 안정성은 유리 효소보다 더 높은 저장온도인 60t에서도 더 안정한 것으로 나타났다. 키틴 고정화 효소에 의한 키토산의 가수분해반응은 반응 3시간까지 급격한 증가를 보이다 그 이후의 반응시간 경과에서도 더 이상 증가를 보이지 않았다. 고정화 효소에 의해 생성된 올리고당의 조성은 효소의 반응시간에 따라 크게 의존하였으며, 2시간의 반 응에서 비교적 고차 올라고당인 COS-4-6의 함량은 약 90% 이상이었다 두 효소에 대한 반용속도상수에서, 고정화 효소는 유리 효소에 비해 낮은 기질친화성과 낮은 반웅속도를 보였지 만, 높은 기질농도에서도 전혀 기질저해반응은 일어나지 않았다. 따라서 키틴 고정화 효소는 유리 효소에 비해 활성의 감소없이 효율적으로 키토산을 가수분해할 수 있었으며, 고차 올리고당의 생성 량도 매우 높았다.

Enzymatic hydrolysis using an immobilized enzyme was carried out to produce chitosan oligosaccharides (COSs) from chitosan effectively. Chitosanase was immobilized on eight different carriers by physical adsorption. The enzyme immobilized on chitin had higher activity than those immobilized on the other carriers in spite of its lower adsorption. The activity of chitin-immobilized enzyme was more than 90% of the original activity. Optimal temperature of the immobilized enzyme increased by about 15℃ and its thermostability was excellent in relatively wide range of temperature. But its effects of pH did not improve compared to the free enzyme. The immobilized enzyme produced 153 mg/g chitosan of the reducing sugar for 3hrs of hydrolytic incubation time. The total content of higher oligomers, tetramer to hexamer, among amount of total COSs obtained for 2hrs was more than 90%. In kinetic parameters for both enzymes, immobilized enzyme showed lower affinity for substrate and reaction rate than free enzyme, however, no reduction of the rate for high substrate concentrations. Consequently, chitin-immobilized could effectively hydrolyse chitosan and produce the higher COSs without activity decrease in comparison with the free enzyme.

The hairy roots of Crotalaria sessiliflora were induced from the tissue segments infected with Agrobacterium rhizogenes ATCC 15834. The induced hairy roots were subjected to paper electrophoresis fro the detection of opine-positive clones which were considered to have been transformed. Mannopine and agropine were presented in hairy root clones while mannopine was presented in two hairy root clones. Eight hairy root clones were selected and cultured in MS, B5 and WP media. Each of hairy root clones was showed a difference in branch pattern and growth rate. The best culture medium and culture conditions of hairy roots were in MS(3% sucrose, pH 5.7) liquid medium at 25℃, 70 rpm under dark, the growth rate in MS liquid medium was increased with 210-fold more than that of inoculated hairy roots and with 2-fold more than that in MS liquid medium. Also, the adequate condition for hairy root growth was such that concentration of KH$2PO$4 was 1.25mM and the ratio of NH4 : NO was 1 to 3 in MS medium. The presence of pyrrolizidine alkaloids, monocrotaline, in the hairy roots was detected by TLC.

The adsorption behaviors of two major cellulase components, endo I and exo II, from Trichoderma viride were investigated using a-celluloses with different correlation crystallinity index(Cc) as substrates. The adsorption of cellulase enzyme components was significantly affected by the reaction condition and the physicochemical properties of the cellulose. The a-cellulose was hydrolyzed in the presence of cellulase for various periods. The correlation crystallinity index of a-cellulose increased with increasing the hydrolysis time. The adsorption was apparently found to obey the first-order kinetics, and the adsorption activation energy(Ea) was calculated from the adsorption rate constant(ka). The value of adsorption rate constant for endo I was larger than that of exo II. This means that endo I are adsorbed more rapidly than exo II. With the increase in correlation crystallinity index, the values of the adsorption rate constants for endo I and exo II decreased, respectively. The activation energy for the adsorption of exo II on the cellulose also was larger than that of endo I. Also adsorption activation energy of endo I and exo II increased with an increase in the crystallinity of sample cellulose.

Gamma irradiated and non-irradiated Agrimonia pilosa Ledebour were extracted by 70% ethanol. The combined effects of the Agrimonia pilosa Ledebour extract and NaCl on survival of Escherichia coli O157:H7 in tryptic soy broth were investigated. E. coli O157:H7 decreased ca 1 log cycle by the addition of 2% sample extract, and the anthbacterial activity was increased as the concentration of sample extract was increased. The irradiation effect of the sample on antibacterial activity was not observed. On the treatment of NaCl alone, E. coli O157:H7 was inactivated (ca 3~4 log cycle reduction within 48 hr) in more than 7% NaCl. The higher inactivation(ca 5 log cycle reduction within 48 hr) occurred in the presence of 2% sample extract and 5% NaCl than in the addition of each alone. The extracted antibacterial substance was stable in the pH range of 4.0 to 7.0, heat treatment at 121℃ for 15 min, and freezing at -18℃ and thawing at 37℃. There fore, the sample extract, would substantially increase the food-safety in terms of E. coli O157:H7.

유럽주목(Taxus baccata Pendula)현탁 세포 배양에서(10-deacetyl) baccatin III 생산을 증진시키는 연구를 하였다. 높은 초기 당 농도가(10-deacetyl) baccain III 생산을 증진시키는 연구를 하였다. 6% 의 초기 포도당과 자당의 경우 1O-deacetyl baccatin III 생산을 각각 3.배와 2.5배씩 증가시켰다. Baccain III도 초기 포도당 8%에서,초기 자당 6%에서 최대값을 보였다. 일리시터로서 methyl jasmonate는(10-deacetyl) baccatin III 의 생성은 59uM에서 최대치를 보였는데 이는 대조구에 비하여 4.5배 증가한 값이었다.Baccatin III 의 경우 methyl jasmonate 100uM에서 생성된 Baccatin III 최대치는 대조구에 비하여 7.배 증가한 값이었다. Methyl jasmonate elicitation 에 의한(1O-deacetyl) baccatin III.과 각 taxane의 시간별 생성 경향을 보면 baccatin III와 10-deacetyl baccatin III는 투여 후 1일 까지 생성이 증가하다 그 후부터 감소하였고, paclitaxel, 10-deacetyl taxol 및 cephalomanine 의 생성이 이어졌다. (10-deacetyI) baccatin III의 생합성을 증가시키기 위하여 전구 물질을 투여하였다. Benzoic acid를 500uM로 투여 하였을 때 10-deacetyl baccatin III 및 baccatin III의 생산은 대조구의 비하여 각각 10배와 13배 증가 하였다. Lysine도 500uM로 투여하였을 때 baccatin III 의 생성량을 대조구 보다 8배 증가시켰다. 증가시켰다.

Enhanced production of (10-deacetyl) baccatin III and related taxanes was observed in suspension cultures of Taxus baccata Pendula. six % of initial glucose and sucrose concentration increased 10-deacetyl baccatin III production 3.5 and 2.5 times, respectively. Methyl jasmonate, as an elicitor, increased taxane production. Time course changes of taxane production after methyl jasmonate addition showed that baccatin II and 10-deacetyl baccatin III were detected first and paclitaxel, 10-deacetyl taxol and cephalomanine were produced in sequence. Feeding experiments with 500uM of benzoic acid increased 10-deacetyl baccatin III production 10 times. Baccatin III production was also increased 8 times by feeding of 500uM of lysine as a precursor

부착의존성 세포주인 Trichoplusia ni 의 유래의 BTI-TN5B1-4 (TN5) 곤충세포주를 이용하여 인간 혈소판생성축진인자인 재조합 인간 트롬보포이에틴(rhTPO)의 배양조건 최적화 연구를 수행하였다. 배양배지, 세포감염에 투입되는 재조합 베큘로바이러스와 숙주세포의 비율(MOI),세포감염시 세포밀도, 배지 회수시간 및 배양방법 등이 rhTPO 의 생산에 미치는 효과를 연구하여 60 mm dish로 정체 배양시 10 MOl 이상, 2x106 cells 의 세포밀도,바이러스 감염 후72 시간에서 rhTP0 의 최대 발현양 (약 12 mg/L)을 나타내었다. 배양 배지로서는 EXCELL FIVE 배지가 SF900II나Insect serum free media-1 Figure 5. Effect of growth phases on rhTPO production. TN5 cells were grown as suspension culture in 1 L spinner flask with 200 mL of SF900II serum free medium at 80 rpm. The cells were infected with AcBac404-2 at MOl of 1. Culture medium was collected at given time intervals and the expression level of rhTPO was analyzed by ELISA (A) or immunoblot analysis (B). Lanes 1 and 7; cell density of 0.6x106 cells/mL, lanes 2 and 8; cell density of 1.6x106 cells/mL, lanes 3 and 9; cell density of 2.0x106 cells/mL, lanes 4 and 10; cell density of 3.0x106 cells/mL, lane M; prestained molecular weight marker (Bio-Rad). Lanes 1, 2, 3, and 4; culture medium was collected at 48 hpi and lanes 7, 8, 9, and 10; culture medium was collected at 72 hpi. Figure 6. Effect of culture media on rhTPO production. TN5 cells grown with different culture media were infected with AcBac-404-2 at 10 MOL 10uL of culture medium was run on SDS-PAGE and Immunoblot analysis was performed. Lane ];TN5 cells cultured with SF900II serum free media(Gibco),and lane 3; TN5 cells cultured with EXPRESS FIVE serum free media (Gibco) 에 비해 더 증가된 발현양을 나타내었다. TN5 세포주를 0.2 L 규모 (1 L spinner flask)oJl에서 세포간의 응집현상 없이 부유배양에 적응,배양시킨 후 세포성장 시기에 따른 발현을 조사한 결과 1 MOI의 감염조건 하에서는 0.6x106cell/mL의 early exponential시기의 세포밀도에서 72시간 배양하였을 대 최대 발현양을 나타내었다. 나타내었다.

In order to obtain high-level production of recombinant human thrombopoietin (rhTPO) in insect cell line, HTI-TN-5B1-4 (TN5), conditions for optimal rhTPO expression such as multiplicity of infection (MOI), the cell density at infection, harvesting time and type of culture method as well as growth media were determined. When TN5 cells were cultured as anchorage-dependent state in 60-mm dish, cell density 2x6 cells,MOI of 10 and Garvesting the culture media at 72 hr post-infection wrere the cinditions for highest rh TPO production. High production of rhTPO was also achieved by using EXPRESS FIVE serum free media rather than SF900II serum free media-1. Anchorage-dependent TN5 cells were adapted as a suspension culture when they were grown in the presence of heparin. TN5 cells were successfully cultured at 0.2 L scale in suspension culture without having aggregation. When TN5 cells were cultured as suspension state, cell density of 0.6x106 cells/mL, MOI of 1 and harvesting the culture media at 72 hr post-infection, gave the highest yield of rhTPO.

For mass production of polyhydroxyalkanoates (PHA), high cell density cultures of Alcaligenes eutrophus by fed-batch culture under phosphate-limitation condition has been investigated. PHA accumulation by the regulation by the regulation of initial phosphate concentration could be automatically induced, and high density cell culture above 200 g/L also could be successfully produced. The production of Poly-B-hydroxybutyrate (PHB) and dry cell weight increased with increasing the initial phosphate concentration. When the initial concentrations of phosphate were in the ranges of 1.5~4.5 g-PO4/L, PHB and dry cell weight obtained were 83~266 g/L and 61~216 g/L, respectively, and PHB productivity was in the ranges of 1.35~3.10 g/L.h. When a mixture of glucose and propionic acid is used as carbon sources, poly(3-hydroxybutyrate-co-poly-3-hydroxyvalerate), P(3HB-co-3HV), could be also successfully produced under phosphate limitation condition. When the mole ratio of propionic acid to glucose in the feeding solution is 0.22, a final dry cell weight of 150 g/L and a P(3-HB-co-3HV) of 90 g/L were produced. Morphological changes and size distribution of PHB granules synthesized in A. eutrophus under phosphate-limitation condition are determined by TEM during the course of fed-batch. Mean granule diameters of PHB produced are in the range of 0.36~0.39 m, and mean cell size was elongated from 0.54~0.59 um 1.3~1.5 m to 0.83~0.89 m 2.0~2.3 m. Phosphate concentration in media did not affect size distribution of PHB granule and cell.

Rhodococcus sp. 3T6-5Mj 균주의 배양액으로부터 0.2um membrane filter을 통과시키고, 아세톤 침전, DEAE-Sephadex A-50 column chromatography의 약음이온 교환수지,cholesterol affinity column chromatography를 통과시켜서 콜레스테롤 산화 효소를 25.6units/mg의 비활성도, 15%의 회수율,88배로 정제할 수 있었다. 본 균주가 생산하는 콜레스테롤산화효소의 분자량은 SDS-PAGE로 측정한 결과 약,52,000 daltons 정도로 추정되었다. 그리고 이 효소의 Km값은 콜레스테롤을 기질로 측정하였을 때3.2x10-4M 이었다. 본 효소의 최적온도는 50℃ 최적 pH는 7.0으로 나타났으며, 열에 대한 안정성은 30~45℃,pH에 대한 안정성은 6.5~11.0으로 나타났다. 본 효소는 cholesterol, campesterol, stigmasterol, hecogenin, Bm-sistosterol의 기질에 특이성을 나타내었다. 본 효소의 아미노산 440개의 잔기로 이루어져 있으며,그 아미노산 조성은 aspararine/aspartate > glutamine/glutamate > proline > alaline > histidine 의 순으로 나타났다. 본 효소는 효소적 혈중 콜레스테롤의 측정방법에 필수적인 콜레스테롤 산화 효소의 이용 가능성을 보여 주었다.용 가능성을 보여 주었다.

The cholesterol oxidase was purified from the culture broth of Rhodococcus sp. 3T6-5Mj strain by procedures involving filtration, acetone precipitation, DEAE-Sephadex A-50, and cholesterol affinity column chromatography with a recovery of 15% to specific activity of 25.6 units/mg. The molecular weight of the enzyme was estimated to be 52,000 daltons by SDS-PAGE. Optimum pH and temperature for the enzyme activity were approximately pH 7.0 and 50℃ respectively. The Michaelis constant (Km) for cholesterol was found to be 3.2x10-4M. The enzyme showed a high substrate specificity for 3B-hydroxysterols and the relative oxidation rates were 100% for cholesterol, 89% for campesterol, 55% for stigmasterol, etc. Amino acid analysis showed that the enzyme protein was composed of 440 amino acid residues without cystein and tryptophan.

We investigated the effects of dissolved oxygen (D.O.) and fructose (C-source) on cell growth and biosynthesis of cyclosporin A (CyA) produced as a secondary metabolite by a wild-type filamentous fungus, Tolypocladium inflatum. This was performed by controlling the level of D.O. and the residual C-source, as required, through adjustment of medium flow rate, medium concentration and agitation rate in fed-batch cultures. CyA production was furned out to be maximal, when D.O. level was controlled around 10% saturated D.O. and concentration of the C-source was maintained sufficiently low (below 2 g/L) not to cause carbon catabolite repression. Under this culture condition, we obtained the highest values of CyA concentration (507.14 mg/L), Qp (2.11 mg CyA/L/hr), Yx/s (0.49 g DCW/g fructose),Yp/s<(22.56 mg CyA/g fructose), and YTEX>$_p/x$ (48.31 mg CyA/g DCW), but relatively lower values of cell concentration (11.98 g DCW/L) and cell productivity (0.043 g DCW/L/hr), in comparison with other parallel fed-batch fermentation conditions. These results implied that, in the carbon-limited culture with 10% saturated D.O. level, the producer microorganism utilized the C-source more efficiently for secondary metabolism.

The characteristics of de-emulsification of pertroleum emulsion by Nocardia amarae were investigated. Insoluble medium containing n-hexadecane was more effective than soluble medium in de-emulsification of emulsion containing diesel and bunker C as the organic phase. Emulsion made by the addition of xanthan or bioemulsifier was de-emulsified by N. amarae, and longer culture age was effective. In low viscosity range, organic phase with longer carbon chain was more effective. The contact, angle between bacterial film and water droplet in air increased from 16 degree for 4 day culture age to 26 degree for 15 day. The contact angle between bacterial film and water droplet in kerosene, n-heyxane or n-hexadecane also increased to greater than 100 degree after 3 day culture age. The hydrophobicity of bactgerial film increased according to the culture age.

Polymerase chain reaction(PCR) was conducted to obtain the gene encoding glucose oxidase(GOD) from Aspergillus niger(ATCC 2110) and the DNA sequence determined was coincided with published GOD sequence from A. niger. Recombinant transforming vector containing GOD and hygromycin B(hyg.B) resistant gene(hph) was constructed and used for further transformation of A. niger ATCC 2110. Selectivity of hyg.B against A. niger differed depending on which media were used i.e., nutrient-rich media such as potato dextrose agar(PDA) and complete medium(CM) showed only 50% growth inhibition at 400 m ml of hyg.B while the minimal media inhibited mycelial growth completely at 200um ml of hyg.B. Twenty to sixty putative transformants were isolated from the hyg.B-containing minimal top agar, transferred successively onto alternating selective and nonselective media for a mitotic stability of hyg.B resistance and, then, single-spored. Among the stable transformants, the transformant(GOD1-6) grown by flask culture showed the considerable increase of extracellular GOD activity, which was estimated to the degree of 50% - 100% comparing to that of wild type. Transformation of tGOD1-6 was resulted from integration of the vectors into heterologous as well as homologous regions of the A. niger genome. Southern blot analysis revealed that there were two independent integrations of vector into fungal genome and one into the GOD gene due to homologous recombination. In addition, GOD activity and sodium gluconate production when tGOD1-6 was fed-batch fermented were enhanced 11 fold and 2.25 fold, respectively, compared to that of the wild type.

페놀수지인 resole을 유일 탄소원으로 분해할 수 있는 능력을 가진 3개의 미생물 컨소시염이 분리되었다. 이들 마생물 컨소시 염은 페놀수지 resole 제조공장 주변의 토양샘플로 부터 유래하였다.그들 컨소시엄 가운데 MS2로 명명된 미생물 컨소시엄은 배양 12일 이내에 초기에 배지내에 주어진 resole (100 mg/L) 의 70%까지 분해되었으나, 완전분해는 이루어지지 않았다 배양 기간동안 pH가 7.0에서 2.7로 감소되었으며 이러한 조건하에서 resole 분해는 억제되었다 UV /vis-spectrophotometer가 잔존 resole의 정량적 측정을 위하여 이용되었으며, 배양가간중에 채 취된 시료에서 resole의 농도는 UV-scans으로 261 nm에서 최대 흡광치룹 토대로 측정되었다.

Three microbial consortia were screened for their ability to degrade phenolic resin, resole as a sole carbon source. These microbial consortia were derived from soil samples collected from a phenolic resin manufacturing plant site. Among the consortia, the test consortium, designated as MS2, displayed approximately 70% degradation of the substrate, 100 mg of resole per liter, within the fist twelve days of incubation but the degradation was inhibited. During the incubation period, pH was decreased from 7.0 to 2.7, and the resole degradation became inhibited under the conditions. UV-scans of spent culture showed that the wavelength of maximum absorption was 261 nm for resole.