Earticle

세포질 유전형질의 도입을 성공적으로 수행코자 세질 웅성 불임(eMS; cytoplasmic male sterili­t ty) 계통의 담배 MS(male sterility) Burley 21의 callus tissue에서 cytoplast를 분리시키고 이와 Ni­c cotiana tabacum Burley 64의 mesophyll protoplast 를 PEG방법에 의한 세포융합으로 cybrid cell올 유도할 수 있었다. cytoplast의 분라는 density gradient solution의 osmolarity를 일정하게 하는 것보다 gradient간의 차이가 더 효과적이였으며 분리된 cytoplast의 양은 재료로 이용된 protoplast의 20-30% 정도를 나타 내었다. 또한 cytoplast와 protoplast의 융합은 P PEG농도 50%에셔 효과적이었다

This study was investigated as a step for the purpose of successful introduction of cytoplasmic inherited characters between the different plants. Cytoplasts were separated from the protoplasts of CMS(cytoplasmic male sterility) line such as MS Burley 21 which carried from Nicotiana megalosiphon. The cytoplasts were fused to protoplasts derived from Nicotiana tabacum Br 64 with PEG(polyethylene g1yco1). The cytoplasts were separated by density gradient centrifugation. Efficient separation of cytoplasts depended on the difference of specific density of gradient solution. However, the iso-osmolality of gradient solution was not important to separate the cytoplasts. The cells for a cybrid were fused with 50% concentration of PEG.

한 개의 pladsmid에 Gus gene과 Hygromycin resistant gene(Hpt)을 합친 vector를 개발하였다. 이 재조합된 DNA를 벼의 건조한 종자에 imbibition시켜 형질전환했을 때, hygromycin 배지에서 선별한 결과 그 발현율은 single Hpt vector와 차이 가 없었으며, 주로 뿌리의 생장점이나 자엽초에서 Gus expression을 볼 수 있였다. 또한 hygromycin 배지에서 선별되어 성체가 된 개체에서 그 genomic DNA를 뽑아 PCR을 한 결과 1Kb Hpt gene을 확인하였다. 그리고 생체에서 추출한 총 RNA에서 cDNA를 만든 후 reverse transcription PCR을 통하여, 외부 유전자의 발현을 증명하였다.

A vector for plant transformation which had two reporter genes(Gus and Hpt genes) in a single plasmid was constructed. After rice embryos imbibed DNA solution, DNA uptake and gene expression in rice were monitored. Main expression sites of the Gus gene were meristem of root and coleoptiles. There was no difference in Hpt gene expression between a single Hpt vector and the constructed vector in viability of rice in the hygromycin medium after DNA imbibition, The genomic DNA and total RNA extracted from rice transformant survived in the hygromycin medium were subjected to PCR and RT PCR analysis, respectively. As a result, we found the existence of the Hpt gene and its expression in rice.

Vinblastine, an anticancer agent was produced by chemical coupling of two different monomeric indole alkaloids, vinblastine and catharanthine in the presence of ferric ion. Vindoline was efficiently extracted from the leaves of vinca (Cafharanthus roseus) by using supercritical carbon dioxide, whereas catharanthine was chemically extracted from the in vitro cultured hairy roots. The extracted crude monomeric precursors were purified by a two-step preparative TLC. The coupling reaction was carried out in the 0.1M glycine buffer(pH 2.0, 5ml) containing 40mM FeC13 with purified vindoline(0.3mg) and catharanthine(0.3mg) at 4℃. The production yields (weight %) of vinblastine and 3', 4'-anhy-drovinblastine in the products were 23.2 and 26.0, respectively. The produced vinblastine was confirmed by FAB-MS.

호르몬이 결여되고 polyamine이 농도별로 처리된 M MS배지에서 10일 동안 발아생장(light, 16hrs: d dark, 8hrs) 시켜서 얻은 벼 잎 절편에서 엽록소 함 량은 polyamine 처리구가 대조구에 비해 높았으며, s spermine(O.OlmM, O.lmM, ImM) 이 가장 효과적이였다. 한편 chloroplast peroxidase의 활성은 polyamine 처리구가 대조구에 비해 전반적으로 높았으며, 특히 1mM spermidine 처리구는 약 100% 정도 활성을 증가시켰다. SDS-PAGE에 의해 chloroplast thylakoid membrane protein의 band를 조사한 결과 polyamine 처리구와 대조구에서 56, 2 25Kd의 major band를 얻었으며 이 band들의 total a area는 polyamine 처리구가 대조구 보다 더 높았다. 이러한 결과들은 벼 유식물에서 엽록체 발달에 p polyamine이 중요한 인자로 작용한 것으로 사료된다

The effect of polyamine on chlorophyll amount, chloroplast peroxidase and chloroplast thylakoid protein in rice leaf segments which were grown for 10 days(16 hrs, light : 8 hrs, dark) in a hormone-free MS medium containing polyamine was studied. Polyamine treatment increased the chlorophyll contents compared with the control in rice leaf segments. Especially spermine was most effective. Also, in rice leaf segments treated with polyamine chloroplast peroxidase activity was higher than in the control. The treatment with lmM of spermidine increased the enzyme activity by 100%. In polyamine treatment and control two major polypeptide bands corresponding to 56 and 25Kd molecular weight were clearly resolved with other minor bands by SDS-PAGE in the insoluble protein fraction. However, in these bands (56, 25Kd), the total area of protein in treating with polyamine were higher than that of the control. These results suggest that polyamine was an important factor in the chloroplast development of rice seedlings.

Taxol은 최근 항암제로 각광을 받고 있는 물질로서 매우 복잡하고 고 기능성을 갖는 diterpene 알칼로이드이다. 본 연구에서는 국내의 덕유산, 소백산, 태백산 및 수원지역에서 서식하고 있는 주목나무들의 부위별 taxol 함량을 조사하였다. 그 결과 소백산에서 채취한 주목나무에서 가장 많은 taxol 양이 분석되었다. 부위별로는 껍질, 잔가지, 잎 순으로 taxol함량이 높게 나타났다.

Taxol is the promising diterpene alkaloid with antineoplastic activities. The taxol content in various parts of yew trees in Korea, Mt. Deokyu, Mt. Sobaek, Mt. Taebaek and Suwon area, have been determined. The highest taxol content was observed in yew trees of Mt. Soback. In various parts of trees the taxol content in bark was highest and the taxol content in twig was higher than that in leaves.

The objective of this study was to develop an artificial dentin for easy handle and accurate observation of the mechanism on dental caries and to screen biologically active materials from the extracts of traditional plants and fruits for prevention of early dental cares. In order to produce disc PAHA (artificial dentin), the powdered hydroxylapatite was immobilized in a 20% polyacrylamide gel. The characteristics of disc PAHA was very similar to the surface, figure and lattice of human enamel. After decalcification in 0.1M citric acid based on observation with SEM. The critical point of decalcification of disc PAHA by acids was found to be pH 5.0-5.5, which was hi agreement with human enamel. The degree of decalcification from disc PAHA in 0.1M citric acid solution was sixfold higher than that of human enamel. This result suggested that disc PAHA would be useful as a substitute of human enamel for in vitro experiment. The extracts of garlic and Flower Apple A, B seemed to inhibit growth of S. mutans. Especially, when the 300ul of its extracts added to the medium to incubate S. mutans, F. apple B showed strongly an inhibitory effect in both the growth of S. mutans and the synthesis of insoluble glucan.

효모균에서의 유전자 재조합에 의한 단백절 생산에 미치는 유전학적, 환경적인 요인의 영향을 연구하였. Plasmid 안정도와 개수는 REP+ 체계 에서 대단히 높은 반면, rep 체계에서는 매우 낮았다. 2um circle plasmid genome을 포함하는 plasmid의 경우에 있어서. [ciro] 형 세포에서의 plasmid 안정도와 개수가 [cir+]형 세포에서보다 높기때문에 [cir+o]형 세포가 더 선호되는 세포였다. 유전자 발현은 plasmid 개수와 안정도에 좌우 되었다. 촉진제의 양이 유전자 발현에 매우 중요 한 역할을 했다. 유전자 발현의 촉진에 필요한 g떠actose의 농도는 0.8% 이 변 충분했다. 높은 안정 도와 개수를 갖는 plasmid의 경우 촉진속도는 매우 빨랐다. Galactose가 배양의 시작부분부터 첨가 될 때가 mid-exponential ph잃e에 첨가될 때보다 유전자 발현의 극대점에 이르는 시간이 걸었다. 상대적 촉진제의 양이 증가함에 따라 glucc잉e억제 현상은 감소되었다

The effects of genetic and environmental factors on productivity of a cloned protein were studied in recombinant Saccharomyces cerevisiae. Plasmid stability and copy level were very high for a REP+ system(at ca. 10 generations, stability: 65-90%, plasmid copy number per cell: 40-200), whereas these were very low for a yep- system(at ca. 10 generations, stability: 30%, plasmid copy number per cell 20). In plasmids containing the 2um circle genome, a[ciro] strain was a preferred host cell since the plasmid stability and the copy number in a [ciro] strain were higher than in a [cir+]strain. Cloned gene expression was dependent on plasmid copy number and stability. The inducer (galactose) level played a very important role in cloned lacZ gene expression, showing that a galactose concentration of 0.8% was sufficient for induction of gene expression. Induction rate was very fast in the case of plasmids exhibiting high stability and copy number by a factor of 4 to 25. The time to reach the peak value of gene expression was longer when galactose was added at the start of fermentation (ca. 26 hours) than at the mid-exponential phase (ca. 6 hours). Glucose repression was reduced by a factor of 2 to 5 as the relative inducer level increased.

Activated carbons were used to examine their performance for the separation of undesirable colored materials from culture broth containing hyaluronic acid. Six local samples and a NORIT ROX 08 were tested, whereas the latter was mainly studied under batch and continuous modes. The optimal wavelength for the detection of colored materials was 330nm. The optimal choice of NORIT ROX 08 provided 30% colored residuals with 96% hyaluronic acid recovery of original broth in batch experiments. The nonlinear adsorption behavior of protein and colored materials with activated carbon (C) was correlated by a Langmuir equation to give 18C/24+C and 500C/892+C for protein and colored materials, respectively. It appears that colored materials were composed of 78% protein and 22% glucose residuals on the basis of clearance results. A microscopic study using a scanning electron microscope suggests that regeneration of used activated carbon with 0.1N NaOH and hot water was not satisfactory. The present study proposes that the continuous monitoring of colored materials during purification can be accomplished by Installation of a UV monitor commonly used for continuous detection of protein during the process, as resulted from the significant correlation of color (A330)=0.353protein(mg/ml)+0.1(R=99.7%).

난분해성인 색소를 대사분해할 수 있는 미생물을 분리하여, 색소분해의 최적조건을 조사하였다. 미생물은 폐수처리공장의 폭기조에서 채취한 폐수로부터 agar media에서 순수분리하였다. 이 균주들을 glu­c cose, peptone, Na2HPO45,KCI,MgSO4,KH2PO4,NaCI,CaCI2와 dye(azo계, reactive red계 ) lOppm 을 함유한 액체 배지에서 dye의 분해력이 강하였으 며, 이때의 최적 pH는 중성 또는 약알칼리성이고, 최적 온도는 30℃ 전후이다. 이 조건에서 10ppm의 m mono-azo(Lot No. 180), di-azo(Lot No. 138), re­a active red(Lot No.2)는 약 2일에 거의 분해되었고, di-azo(Lot No. 151), reactive red(Lot No. 34, L Lot No. 00166)는 약 5일에 거의 분해되었고, 최적 배지조건하에서 산소의 영향은 D.O를 50%로 유지 시켰을 경우보다 혐기성 배양의 경우가 reactive d dye(Lot No.2)의 분해도가 훨씬 높았다.

This study describes biodegradation of dyes which are used in textile industries. Dyes released into the environment from industrial waste water are considered to be a serious pollution problem because of the wide spread into environment with a variety of colors. The microorganisms used in this experiment were Pseudomonas species, which had been screened from aeration tank of waste water treatment. It was found that optimum concentrations for culture media were 14g of glucose, 6g of peptone, 160 mg of Na2HPO4, 200mg of KCl, 140mg of MgSO4,.7H2O,1.0g of KH2PO4, 400mg of NaCl, 200mg of CaCl2 and dye 10ppm per litre of distilled water. The high efficiency of dye degradation was obtained at pH 7-8 and 30-35℃. Strains screened are excellent for removal of azo and reactive dyes, which are relatively stable and difficult to degrade. Dyes of 10ppm such as mono-azo (Lot No. 180), di-azo (Lot No. 138) and reactive red(Lot No. 2) were mostly decolorlzed within 2 days and di-azo (Lot No. 151) and reactive red(Lot No. 34, No. 00166) were decolorized within 5 days in the controlled fermenter. In the case of reactive dyes, oxygen supplies showed lower biodegradability compared to anaerobic culture.

Polyester 감량폐수의 주성분인 EG,TPA분해균을 분리하여 각각 Pseudomonas sp.로 동정하여 Pseudomonas sp. EAW, Pseudomonas sp. TS2로 명명하였다. 이 두 균주의 최적 배양조건은 pH 7.5, 온도 35℃ 및 질소원은 (NH4)2SO4 in EAW의 경우 niacin과 biotin, Strain TS2의 경우 Na2MoO4ㆍ2H2O와 thiamine이 각 균주의 성장과 분해율에 약간씩 영향을 미치는 것으로 나타났다. Strain EAW는 접종량의 증가에 따라서 성장 및 EG의 분해시간이 현저히 감소하였으나 Strain TS2의 경우는 접종량이 증가하여도 성장 및 분해율이 큰 차이가 없었다. 또한 rich-medium에 계대배양한 각 균을 EG, T TPA액체배지에 접종하였을 때 계대배양의 회수가 증가함에 따라 Strain EAW가 Strain TS2보다 성장 및 분해율이 현저히 감소하였다. 실제 폐수에서 균들의 성장 및 분해율이 분리용 배지의 경우 보다는 약간 감소하였으며 EG, TPA 는 배양 48hr 후 CODMn and CODcr의 제거율은 89% 와 93%로 각각 나타났다. 회분배양시 EG,TPA의 초기농도가 25g/L 이상에서는 각 균주의 비성장속도가 감소하였다.

Strains degrading ethylene glycol(EG) and terephthalic acid(TPA) were isolated from water systems, and identified as Pseudomonas sp. They were named as Pseudomonas sp. EAW for EG and as Pseudomonas sp. TS2 for TPA. The optimal culture conditions of temperature, pH and nitrogen source were found to be 35℃, 7.5 and ammonium sulfate, respectively. The growth of strains and removal efficiency was slightly promoted by trace elements such as niacin and biotin in case of EG, and by trace elements such as Na2MoO4ㆍ2H2O and thiamin i case of TPA. With increasing inoculation sloe for batch culture, the removal efficiency of EG by the strain EAW was conspicuously increased, while the removal efficiency of TPA by the strain TS2 was not changed as much as that of EG. The growth rate of the strain EAW was much more decreased than that of the strain TS2 in the enrichment medium, as the frequency of repeated-batch culture in the rich-medium increased. in case of real wastewater, growth rate and removal efficiencies of EG and TPA were lower than those in the enrichment medium. CODMn and CODcr removal efficiencies after 48 hrs batch culture in real wastewater were 89% and 93%, respectively. The specific growth rate was inhibited when the initial concentration of EG or TPA was more than 25g/L.

Methylomonas mucosa를 이용하여 세포외 다당류의 생산을 알아보았다. 본 미생물은 성장을 위해 메탄올을 탄소원으로 이용하여 세포외 다당류를 생산한다. 효과적인 발효 공정 시스템의 개발올 위해 회 분식, 유가식, 연속식 배양을 수행하여 각각의 당류 에 대한 생산성을 알아보았다. 플라스크 배양에서 메탄올이 약 1 % (v/v) 이상에서 다당류의 생산과 세포의 성장은 저해되었다. 1% (v/v)에서 최대 비성장속도를 나타내었고 C/N 비율이 높을수록 다당류의 생산은 많았다. 다당류의 생산에 Mg2+이온 농도가 크게 영향을 줌을 확인할 수있었다. 유가식 배양에서는 다당류의 농도가 회분식에 비 해 4배 이상 높았으나 수율은 오히려 감소하였다. 연속주입을 통한 유가식 배양은 간헐적 주업방법보다 생산성이 높았다. 이는 간헐적 주입에서 나타날 수 있는 메탄올에 의한 제한이나 저해가 없었기 때 문으로 보여진다. 연속조엽은 산소 제한을 피하기 위해 순수산소를 공급하였다. 희석률이 O.21h-1까지 증가하여도 수융 과 생산성은 증가하였다. 생산된 다당류 용액의 점도는 다당류의 증가에 따라 지수적으로 증가하였다

The production of extracellular polysaccharide by Methylomonas mucosa (NRRL B-5696) was investigated. The microorganism uses methanol as the carbon source for their growth and produces exopolysaccharides. The productivity of exopolysaccharides was investigated under various culture modes: batch, fed-batch and continuous culture. In flask culture the growth of cell mass and the production of polysaccharide were inhibited at above 1% (v/v) methanol. At 1%(v/v) methanol maximum specific growth rate was obtained. As C/N ratio (g methanol/g ammonium sulfate) increased, polysaccharide production increased and cells mass decreased. Magnesium ion was also found to be essential for the polysaccharide production. In batch culture the production of polysaccharides was more affected by the specific growth rate than the cell concentration. In fed-batch culture the concentration of polysaccharide was 4 times higher than that of batch culture, but the yield was lower. The productivity of fed-batch with continuous feeding was higher than that of batch or fed-batch with intermittent feeding. This is due to no methanol limitation or inhibition that used to occur in fed-batch culture with intermittent feeding. In continuous culture pure oxygen was supplied to avoid the oxygen limitation. As the dilution rate in- creased up to 0.21 h-1, the yield and productivity increased. The solution viscosity of the produced polysaccharide obtained from above increased exponentially with the concentration of polysaccharide.

심각한 수질환경 오염을 억제하기 위하여 알코올 발효고정의 폐액을 미생물을 이용하여 BOD를 낮추고 동시에, 단세포 지질의 생산 공정을 개발하고자 하였다. 알코올 발효공장 두 곳의 폐액을 수집하여 그 조성 및 일반성분과 생물학적 산소 요구량(BOD), 화학적 산소요구량(COD) 등을 측정하였고, 폐액중에 생존하는 미생물들과 유지 함량이 높다고 알려진 효모 균주들을 배양하여 단세포 지질의 생산에 적합한 Rhodotorula glutinis 균주를 선발하였다. 알코올 발효 공장의 폐액을 기질로 사용하기 위하여, 전처리 조건을 실험한 결과 NaOH의 처리가 가장 우수하였다. 최대의 지질수율을 얻기 위한 최적 배양 조건을 구한 결과, 배양 온도 30℃, 초기 pH5.0이었고, 인산염의 첨가 효가가 있었다. 발효조를 이용한 단세포 지질 생산을 시도하여 기질 중에 탄소원대 질소원의 비에 따라 단세포 지방산 조성 및 함량에 차이가 있었으며, 탄소원 대 질소원의 비가 50에서 최대 지질계수를 나타내었다. 최종적으로 효모 배양에 의한 단세포 지질이 4g/L 생산되었고, 처리폐액의 BOD의 88.7%가 감소되었다

In order to reduce BOD of wastewater from alcohol distillery manufactory, the production of single cell lipid was attempted. Among five yeast strains tested, Rhodotorula glutinis was most desirable for lipid production. Wastewater was treated with 2N NaOH and used as a medium. The optimum pH and temperature for lipid production were found to be 5.0 and 30℃, respectively. The addition of monobasic phosphate was good for cultivation of Rhodotorula glutinis. The C/N ratio was an important factor for lipid production and composition. The best C/N ratio was 50 for the production of single cell lipid. By cultivation Rhodctorula glutinis for 4 days, 4g/L of single cell lipid was harvested and BOD of wastewater reduced by 88.7%.

Extractive fermentations with the extract of Jerusalem artichoke in an aqueous-two-phase-system of polyethyleneglycol(PEG) and dextran(Dx) were investigated to obtain the effects of composition of PEG and Dx on both fermentation ,characteristics and partition ratio of alcohol. The specific growth rate of K. Fraglis CBS 1555 increased with a decrease of concentration of PEG and Dx. It augmented along with concentration of initial sugar up to 80g/l but decreased thereafter. The specific production rate of alcohol showed a rising tendency up to 100g/lof initial sugar, whereafter represented a decreasing trend. The partition ratio of alcohol between two phases augmented according to decrease of Dx comic. and increase of PEG cone. regardless of initial sugar concentrations. The ratio, however, decreased with Increment of initial sugar concentration at constant composition of PEG and Dx. The partition coefficient of alcohol had an ascending effect to the increase of PEG cone, but it had little effect on the changes of concentrations of Dx and initial sugar. The present study suggests that the optimum composition of PEG and Dx in the aqueous-two-phase-system by extractive fermentation were around 6.5%(w/v) of PEG and 3%(w/v) of Dx in considerations of emulsion state, sedimentation and separation of two phases, alcohol partition ratio, and specific growth rate.

산소발생능에 있어 오랜 기간 안정성을 유지하는 세포와 분리 틸라코이드 막을 단세포성 남조류인 Synechococcus PCC7002로부터 PVA로 고정화하여 구할 수 있었다. 분리 틸라코이드 막 및 고정화한 세포나 틸라코이드 막의 흡수스펙트럼의 굽대가 청색쪽으로 3nm정도 전이하였다. PVA에 고정화한 세포나 틸라코이드 막은 4℃에 보관할 경우 광합성에 의한 전자전달 활성, 특히 광계 2의 활성을 오랜기간 보존할 수 있었다. 세포를 고온에 처리하였을 때 Fo와 Fv가 증가하였으나 고정화한 후에는 Fo의 변화가 거의 없었다. 이 결과는 고정화가 열처리에 의한 광계 2 복합체의 손상 특히 물분해기구의 손상에 대해 보호작용을 할 수 있음을 시사한다.

Highly stable O2-evolving cells and thylakoid membranes have been obtained from the cyanobacterium, Synechococcus PCC7002, by immobilization with polyvinylalcohol(PVA). The absorption peak showed the blue-shift of about 3 nm after immobilization of intact cells and thylakoid membranes as well as isolation of thylakoid membranes. Photosynthetic electron transport activities, especially PS II activity showed greater stability in the PVA-immobilized cells and thylakoid membranes when stored at 4℃ than in those at 25℃. When the cells were threated at higher temperature, the level of Fo and Fv increased. After imobilization, however, Fo showed no change. This suggests that the immobilization can protect against the damages of PS II complex, especially a water-spiliting system, by heat treatment.