Earticle

To produce economically important indole alkaloids by cell cultures, we selected protoplastsderived clones (protoclones) of vinca (Catharanthus roseus) for high yields of catharanthine and ajmalicine. Protoplasts were enzymatically isolated from suspension-cultured cells. The highest plating efficiency (1%) was obtained when protoplasts were plated at a density of 1105 protoplasts/ml in a culture medium solidified with 0.4% Seaplaque agarose. The growth rates of 40 protoclones subcultured on a solid medium varied over a wide range. Protoclone VPC-6, which had the highest growth rate, was observed to produce relatively high yields of catharanthine and ajmalicine when cultured in a liquid medium. Although the original cell line did not produce catharanthine at a detectable level by HPLC, protoclone VPC-10 produced it at a level of 5.9ug/g fresh weight of cells for 10 days of culture. Under the same conditions, protoclone VPC-15 produced ajmalicine at a level of 133.6ug/g, of which productivity was improved about ,3 times than that of the original cell line. The results indicate that differences in the growth rate and indole alkaloid yield among the protoclones reflect the somaclonal variation in suspnsion-cultured cells

We studied the production and excretion of alpha-interferon in recombinant Escherichia coli harboring plasmid pIF-III-B, which carries alpha-interferon gene under the control of lipoprotein and lacUV5 promoter, and lac operator. Basically, the effects of concentrations of ampicillin and an inducer, IPTG, for the expression of the cloned gene, on the productions of alpha-interferon and plasmid stability were studied. The highest production of alpha-interferon was observed at 50 mg/1 of ampicillin concentration and 0.5 mM of IPTG. The plasmid pIF-III-B was maintained very stably in medium with ampicillin but segregated rapidly in medium without ampicillin. Also, the plasmid was segregated more rapidly in medium with an inducer higher than 0.5 mM.

The optimal glucose concentration for the high-density culture of recombinant yeasts was obtained using dynamic simulation. An adaptive and predictive algoritilm complimented by the rule base was proposed for the control of the fed-batch fermentation process. The measurement of process variables has relatively long sampling period and relatively long time delay characteristics. As one of the solution on these problems, prediction techniques and rule bases were added to a classical recursive identification and control algorithm. Rule bases were used in the determination of control input considering the difference between the predicted value and the measured value. A mathelnatical model was used in the estimation and interpretation of the changes of state variables and parameters. Better performances were obtained by employing the control algorithm proposed in the present study compared to the conventional adaptive control method.

감자(Solannum tuberosum) 괴경에 A. rhizogenes를 접종하여 암소에서 배양한 결과 2-4주 후에 hairy root가 유도되었다. 유도된 hairy root는 opine 분석을 통해 agropine과 manopine이 검출되어 형질이 전환되었음을 확인하였으며, 발생이 진행됨에 따라 단백질은 전차 감소 되었고, 전분은 점차 증가함을 보였으며, GS.II의 활성 또한 발생단계에 따라 증가함을 보였다. Ca2+ channel blocker 인 verapamil 처리구에서는 약 60% 정도 GS II의 활성이 감소 되었으며, calmodulin inhibitor인 chloropromazine 처리구에서는 GS II의 활성에 별 영향을 미치지 않았다. 한편 peroxidase 활성은 3주 동안 배양한 callus에서 가장 높았으며, peroxidase 양상은 hairy root에서는 pI 5.4, 5.9의 band가 나타난 반면, 3주 동안 배양한 callus에서는 pI 6.4, 7.1, 7.4의 새롱룬 band를 얻었다

Hairy root induced by A. rhizogenes from potato tuber (Solanm tuberosum L.) synthesized the agropine and mannopine which were demonstrated with paper electrophoresis. And the starch contents in hairy root were increased gradually following the developmental stage. But protein contents were decreased. The activity of B-glucan synthetase II(GSII) which is related to the cell wall biosynthesis was stimulated in hairy root on the developmental stage. And chloropromazine did not influence the activity of GS II while verapamil inhibited about 60% of the activity GS II. Therefore, these results showed Ca2+ to be effective factor in the cell wall formation. Isozyme pattern of peroxidase was investigated in the callus and hairy root induced from potato tuber

The production of poly-B-hydroxybutyrate(PHB) from methanol by batch and fed-batch cultivations of Methylobacterium sp. GL-10 was studied. PHB accumulation was stimulated by the nutrients deficiency including, NH4+, SO42-, and K+. The nitrogen deficiency was the most critical factor for PHB accumulation. In batch cultivation, the maximum cell concentration and PHB content were 1.86g/l and 0.62g/l, respectively, with 1.0%(v/v) of methanol and 0.5g/1 of ammonium sulfate. The mass doubling time of Methylobacterum sp. GL-10 was in the range of 4-5 hrs. The cell growth and PHB accumulation were severely inhibited at the methanol concentration over than 2% (v/v). To overcome methanol Inhibition, constant feeding and intermittent feedillg fed-batch cultivations were adopted, using C/N molar ratio as a control factor. In constant feeding fed-batch process, cell concentration was increased up to 2.67g/1, and PHB yield was enhanced from 0.33 of batch culture to 0.53. The relatively low cell concentration was caused by methanol accumulated in culture broth at late growth phase. To prevent methanol accumulation and to maximize PHB production, DO-state intermittent fed-batch cultivation was attempted. The cell and PHB concentration was reached up to 4.55g/1 and 1.80g/1, respectively. It was possible to maintain methanol concentration low and also to feed nutrient of desired C/N molar ratio.

The aims of this study were investigated the antitumor effects and immunological activities of lipopolysaccharides (LPS) extracted from Proteus vulgaris RH-90 toward sarcoma-180 cells. LPS extracted from Proteus vulgris RH-90 was irradiated with gamma ray for detoxification. The tumor incidence of sarcoma-180 occurs all group which injected with gamma ray irradiated LPS and tumor of sarcoma-180 was necrotized with breeding in the injected group of l0ug LPS. The inhibition ratio of tumor growth showed at the highest level of 60.88% when 5ug gamma ray irradiated LPS was injected into mice. The prolongation ratio of life showed 20.72% when injected into mice with gamma ray irradiated LPS of 5ug. In the effect of immunological activity, the number of circurating leucocyte and peritoneal exudate cells were increased significantly in the treatment group than that control group, and dose-dependent response indicated by the increase of weights of immunorgans which revealed the improvement of immunity. The effect of macrophage on phagocytes, there were not found the differences between phagocytic and corrected phagocytic index.

The objective of this in vitro study is to screen a possible inhibitor, originated from some chinese herb medicines, of 3-hydroxy-3-methylglutaryl Coenzyme A (HMG-CoA) reductase that is the major regulatory enzyme of hepatic cholesterol biosynthesis. Fourteen kinds of herbal plant were extracted with water and evaporated for prescreening. The methanol extracts of the effective 3 kinds (9 species) were fractionated with chloroform, ethylacetate, butanol and water, and vacuum evaporated. The degree of inhibition of the extracts to HMG-CoA reductase activity was calculated by the spectrophotometric method using microsomal protein of Saccharomyces cerevisiae ATCC 42949 as an enzyme source. Among these samples, marked inhibitory effects were observed in the extracts of ethylacetate and chloroform fractions of the Rosa rugosa roots, and those of butanol, ehtylacetate and water fractions of pine leaves. Also, the inhibitory effects of the extracts obtained from buckwheat shell and the roots of Rosaceae were found.

본 연구에서는 L-phcn ylalaninc을 생산하는 조절기작을 상실한 영양요구성 변이주인 Corynebacterium glulamicum ATCC 21674를 이용하여 플라스크내에서의 회분식 배양시의 특성을 조사하였다. 이 균주는 회분반효시 2.1-3.4 g/I 의 phcnllalanine파 2.9-4.4 g/I 의 tyrosine을 생산하였고, 당농도가 높을 경우 생산성이 저하됨을 알 수 있었다. 또한 온도의 변화는 이들 아미노산 생산에 큰 영향을 미침이 관찰되었다. 30℃에사 배양하는 경우, 37℃에서 배양하는 것보다 훨씬 많은 아미노산이 생산되었다. 배양도즙 pH는 급격한 변화를 보였다, 이 균주는 tyrosine이 없는 최소배지에서도 자라는 것이 확인되었고, tyrosine를 과량 생성까시 함으로 보아 영양 요구 성질을 상실한 revertant로서 조전기작 상실성을 ­유지한 것으로 판단된다

An auxotrophic regulatory mutant of Corynebacterium glutamicum ATCC 21674 produced 2.1-3.4g/1 of phenylalanine with 2.9-4.4g/l of tyrosine in the batch shake flask fermentations. At higher sugar concentration, the production of both amino acids was lower than that at low sugar concentration. There was a pronounced effect of temperature on the amino acid production. At 30℃, much higher levels of phenylalanine and tyrosine were produced than those at 37℃. The pH decrease in the shake flask fermentation was so fast that it was impossible to maintain a constant pH with calcium carbonate as a buffering agent. Even though the strains we have used are reported as tyrosine auxotrophs, they produced tyrosine and were able to grow on the minimal medium where no tyrosine was present.

When S. fradiae was cultured in S medium, it stavted to produce neomycin in the middle of stationary phase of growth. Antibitoic production is regulated not only by glucose but also by metabolites formed from glucose. A chemically defined minimal salt broth was developen for the study of metabolites activating produition of antibiotic in a neomycin producer. When growth and production or antibiotic in minimal salt broth was examined with a full grown or a vefctativc mycelium, the medium was found not to be good for the growth, but to be good enough for the production of antibiotic with a full grown mycelium. When many carbotlydrates, organic acids, or alcohol were supplmented with instead of glucose in the medium suspcndcn with a full grown mycelium, the amount of antibiotic produced in the medium containing fumaratc was 5 times more than that in the medium with glucose. Further study indicated that the medium is not good also for the growth but good for the production of antibiotic. The antibiotic produced in this medium was identified to be neomycin. The activation of the production of neomycin by fumarate was further confirmed in a complex medium. Fuinarate is suspected to initiate and to activate the biosynthesis of neomycin at the gene level.

생물고분자인 PHB로 고분자 matrix를 제조하여 대상약물인 silver sulfadiazine의 방출기전을 연구하였다. 고분자 matrix내의 PHB의 함유량 증가와 matrix의 두께비가 증가할수록 방출속도는 늦어졌다. 그러나 가소제량이 증가함에 따라 방출 기전은 각각 18, 17, 16, 12 및 10시간으로 감소하였다. 이 제형은 약물방출기전은 Higuchi model에 따른 확산으로 생각되었다. 결론적으로 PHB의 함유량과 가소제의 함유량 및 matrix 두께의 상관관계를 조절하므로써 특정한 약물의 방출기전을 얻을 수 있는 maxtrx형태의 개발 가능성을 보였다.

Using PHB biopolymer as polymer matrix, the release mechanism of a model drug, silver sulfadiazine was studied. The release behavior actually conformed to the Higuchi's diffusion controlled model. The release rate was delayed with an increasing proportion of PHB, whereas decreased as glycerine concentration incresed. The release rate was increased as the polymer matrix thickness increased.

Experiments of alcohol fermentation of the yeast,K. fragi1is CBS 1555 were performed to obtain the following results. In these experiments, the initial concentrations of sugar which was composed of inulin and fructose as weight ratio of one to one were 30, 50, 75, 100 and 150g/l and the initial densities of the microorganism were less than 0.5g/l, 10g/1 to 15g/1, and 50g/l. The functional relationship among specific growthrates, sugar concentrations, and alcohol concentrations could be expressed by Aiba-shoda equation and the specific growth rate represented the trend that decreased with increase in the initial concentration of the microorganism. Also, umax and Ks of Monod's equation could be expressed as the function of initial cell concentration like the following equations. umax=0.8-0.008X Ks=0.54X+8 In the region that sugar, alcohol and cell concentrations were 10g/1 to 120g/l, 0g/l to 60g/l and 0.5g/l to 50g/l respectively, the differences between the experimental values and the calculated ones for specific growth rate approached to 40% with respect to experimental values at the worst cases, but in most cases, those were distributed in the range of less than 20%.

The media compositions and culture conditions were optimized for mycelial growth of Coriolus versicolor and Lentinus edodes. Media composition for optimal growth of Coriolus versicolor was 2.0% glucose 0.4% peptone and 0.6% yeast extract. Media composition for optimal growth of Lenttnus edodes was 2.0% glucose 2.0% starch 0.4% bacto-soytone and 0.6% yeast extract. The media supplemented with KH2PO4, 0.046% KH2PO4 0.1% and MgSO4, .7H2O 0.05% supported better mycelial growth than the media without mineral salts. The optimum temperature for mycelial growth ranged from 25℃-28℃. The optimum pH range for mycelial growth of Coriolus versicolor was 5.2~5.6 while that of Lentinus edodes appeared to be 5.75.

본 연구는 혼합 반응기에서 파라-톨루엔 설폰산(PTSA) 용액으로 이소부틸렌의 최적 흡수 속도론과 최적 흡수 조건을 연구하였다. 본 실험에서 행한 실험 조건은 온도 20-80℃, 혼합속도 400-1000 rpm, 산농도 30-70 wt% 그리고 산부하(acid loading) 0.3-5 이었다. 결과적으로 최적 조건에서의 이소부틸렌의 흡수속도는 이소부틸렌 농도항에 대한 1차 반응이며 PTSA 농도항에 대해서는 반응 차수가 변한다. 그때의 겉보기 활성에너지는 13Kcal/mol 이었다

The optimum conditions and rates of absorption of isobutylene in aqueous para-toluene sulfonic acid(PTSA) were studied experimentally in a stirred reactor. The effects of acid concentration(30-70 wt%), acid loading (0.3-5), mixing intensity(400-1000 rpm), and temperature (20-80℃) on the absorption conditions were examined. The absorption rate of isobutylene was first order in isobutylene and variable order in PTSA concentrations. The apparent energy of activation was found to be 13 Kcal/mole.

역미셀을 이용하여 박테리아 (Bacillus licheniformis)의 발효액으로부터 알카리성 단백질 분해효소를 분리할 때 여러가지 인자들, 즉 이온강도, pH, 계면활성제의 농도 등이 분리효율에 미치는 영향에 대하여 다음과 같은 결과를 얻었다. 이온강도를 높여 주기 위하여 KCI을 사용하여 조절하였으며, 발효액의 이온강도를 높여줄수록 추출되는 효소의 양은 감소하였으며, 역추출 용액의 이온강도를 높여줄수록 역추출되는 효소의 양은 증가하였다. 계면활성제로는 sodium-di-2-ethylhexyt sulfosuce-inate (Aerosol-OT또는 AOT)를 사용하였으며, 유기상내의 계면활성제 농도를 높여줄수록 더 많은 양의 효소가 추출됨을 알 수 있다. pH는 IN HCI과 0.1N NaOH를 사용하여 조절하였고, 발효액의 pH가 5.3이었을 때 가장 많은 양의 변성되지 않은 효소가 추출되었으며, 역추출 용액의 pH가 7.5일 때 가장 많은 양의 변성되지 않은 효소가 역추출됨을 알 수 있다.

In separating alkaline protease from the bacteria (Bacillus licheniformis) fermentation broth using reverse micelle, effects of various factors;ionic strength, pH and surfactant concentration, on separation efficiency were studied. KCl controls the ionic strength. The lower KCl concentration was in the feed solution, the more protein and activity was recovered. The higher KCl concentration was in the stripping solution, the more protein and activity was recovered. Using sodium-di-2-ethylhexyl sulfosuccinate(Aerosol-OT or AOT) as a surfactant, the higher AOT concentration in the solvent, the more activity and protein were recovered. 0.1N NaOH and IN HCl were used to adjust pH. Maximum recovery of protein mass and activity were obtained at feed solution of pH 5.3. Maximum activity was recovered at stripping solution of pH 7.5

대장균의 plasmid상의 B-lactamase를 IPTG induction으로 대량 생산할 때 precursor processing inhibitor (CCCP)를 가하여 B-lactamase의 soluble fraction과 insoluble fraction (inclusion body)의 생산성을 비교하였다. CCCP로 처리한 경우가 더 많은 soluble B-lactamase를 생성하였으나,inclusion body의 양에는 큰 차이가 없었다. 이것은 B-lactamase precursor processing 속도를 낮출 경우 soluble B-lactamase가 더 많이 생성된다는 것을 보여주었다

The effects of the precursor processing inhibitor, carbonylcyanide-chlorophenyl hydrazone(CCCP), are investigated on the production of soluble B-lactamase the formation of the inclusion body when B-lactamase is overproduced by induction with isopropyl thiogalactoside(IPTG). When cells are treated by CCCP, more soluble B-lactamase is produced. In this case, no difference in the amount of inclusion body is observed.