Earticle

Using the shuttle vector pECCGl between Escherichia coli and Corynebacterium glutamicum and C. glutamicum strain JS231 grown in the medium supplemented with penicillin-G, which inhibits the formation of cross-links in the peptidoglycan of bacterial cell wall, various parameters involved in electroporation system including resistance, electric field strength, capacitance, DNA concentration, and cell density were investigated independently and optimized for the high efficiency transformation of coryneform bacteria. Using cells grown with 0.3U/ml of penicillin-G and harvested at A600 of 0.7-0.8, transformation efficiencies of 107-l08 transformants/ug of DNA with Corynebcctertum glutamicum strain JS231 and wild type ATCC13032 were achieved under conditions of 12.5kV/cm of electric field strength, 400 ohms of resistance, 25uF of capacitance, 3108 cells per transformation(1.21010 cells/ml) and 100ng of plasmid DNA per transformation.

Recently, developments of large scale and high density cell culture methods have been the objects of many researches, because the demand of various pharmaceutical products produced by animal cell culture has been rapidly increasing. The cell culture equipment should have the requirements such as sufficient oxygen transfer and mixing, low shear stress and surface tension, and small foaming. In order to develop a proper bioreactor meeting these requirements simultaneously, a perfluorocarbon having high solubility of oxygen was sprayed into the medium as an oxygen carrier instead of air. Also, a new impeller was developed and combined together with the perfluorocarbon spraying system so as to design a new bioreartor for cell cultivation. The new impeller had better characteristics of mixing and oxygen transfer than the paddle and cell-lift impellers based on the same, shear rate. But, it was observed that the volumetric oxygen transfer coefficient of the new bioreactor decreased with increasing cell density during E. coli fermentation.

본 연구에서는 고정전달자를 갖는 poly(4-vinylpyridine-co-styrene) 고분자막을 합성하여 이를 특성화하고, CI- 및 CCI3COO 의 H+ 및 OH- 농도차에 따른 능동전달 기구에 대하여 고찰하였다. 시험 범위 내에서 CI-의 능동전달은 주로 농도차를 구동력으로 한 병류수송(symprot)이 주가 되었으며 반면에 CCI3COO의 능동전달은 OH-의 농도차에 의한 향류 수성(antiport)의 영향이 큼을 알 수 있었다. 농도와 농도에 의한 음이온의 초기플럭스를 관찰함으로서, 고정전달자와 이온간의 엄형성계수, K와 이온의 막내 확산계수, D는 CI-의 경우 각각 4.60x102 mo-1cm3 및 1.57x103cm2/h, CCI3COO- 의 경우 각각 1.10x104 mol-1cm3 및 1.44x104cm2/h 이었다.

Poly (4-vinyipyridine-co-styrene) membrane containing Pyridine as fixed carrier was synthesized and characterized. And the active transport mechanism of Cl- and CCI3COO- with changing concentration of H+ and OH- was investigated. CCI3COO- was transported not only by a symport mechanism with transfer but also by an antiport mechanism with transfer, while CI- was transported only by a symport mechanism with transfer. Observing the initial flux of anions, salt formation constant between ions and membrane (K), and diffusion coefficient in membrane (D) were calculated as follows: for $C1-, K=4.60x102 mol-1cm3, D=1.57x10-3cm2/h$ and for CCI3COO-, K=1.10x104 mol-1cm3, D=1.14x10-34cm2/h.

Permeabilization of Zymomonas mobilis with CTAB(Cetyltrimethylammoniumbromide) was investigated in order to obtain stable process for sorbitol production in the immobilized system. The optimum conditions for sorbitol formation were obtained in the case of using cells treated with 0.2% CTAB at 4℃for 10 min. Permeabilized cells were treated with glutaraldehyde to cross-link the internal enzyme for the improvement of the enzyme stability. In this way, no significant loss of enzyme activity was apparent during 30-day operation in a continuous process. The productivity of the continuous process at dilution rate 0.2h-1 was 6.51g/1/h for sorbitol. The CTAB permeabilized cells could be used to produce sorbitol in the long term continuous process

The effect of concentration of yeast extract and NH4Cl in the mediun of alcohol fermentation of S. cerevisiae ATCC 24858 on the fermentation characteristics, specific growth rate, sugar conversion, alcohol productivity was experimentally investigated. Regardless of initial sugar concentrations, the values of the above three characteristics increased with augument of concentration of yeast extract. However, the increasing tendency ceased above a certain concentration. The concentration of NH4Cl had little effect on the change of the three characteristics. The functional relationships between the concentration of yeast extract and the characteristics were different according to the initial sugar concentrations, but those between the ratio of yeast extract concentration to initial sugar concentration and the characteristics could be expressed as same forms respectively regardless of initial sugar concentrations. Also the values of the three characteristics approached to the maximum values around 0.085 of the ratio, but did not increase any more above 0.1 of the ratio. We have come to conclusion that the optimum ratio of the yeast extract concentration to the initial sugar concentration was about 0.085 and the ratio should not be decided as greater than 0.1 in the medium of alcohol fermentation of S. cerevisiae ATCC 24858.

The optimal cell concentration and dilution rate for maximum ethanol productivity were obtained using dynamic simulation in cell recycled continuous bioreactor. The good control performance was observed using rule-based STR (self-tuning regulator) compared to conventional STR. Rule-base contained the scheme to implement the STR in an efficient on-off way and the scheme for the controlled variable to reach the optimal value in a short time. Since a mathematical model was used to analyze and estimate the changes of the state variables and the parameters, it was possible to understand the physical meaning of the system

Phosphatidylglycerol(PG) and two unnatural phospholipids, phosphatidylethyleneglycol (PEG) and phosphatidylpropyleneglycol(PPG), were synthesized from ovolecithin using cabbage phospholipase D(PLD) in a emulsion system. Optimum pH and temperature for the enzymatic synthesis of PG, PEG and PPG in the emulsion system was 5.0-5.6 and 37℃, respectively. The maximum activity for transphosphatidylation was obtained with 30-80 mM Ca++. Addition of 25% glycerol was required to convert completely ovolecithin to PG, whereas 16% glycerol was sufficient to attain the highest rate of conversion for both PEG and PPG syntheses, the highest conversion rate was obtained with addition of either 10% ethyleneglycol or propyleneglycol. However, the concentration of alcoholic acceptor should be increased up to 20% to improve selectivity up to 100% for PEG or PPG synthesis. Identification of PEG and PPG was made by analyzing the polyvalent alcohols released after their hydrolysis by HCl or PLD.

This research was carried to investigate the effects of several organic solvents on the enzymatic transphosphatidylation in emulsion and two-phase solvent systems. The solvents having a similar dielectric constant with diethylether were effective for the enzyme activity. Diethylether and butylacetate were the most effective solvents, when added 12-15%(v/v) and 10-40%(v/v), respectively, for the synthesis of phosphatidylglycerol, phosphatidylethyleneglycol and phosphatidylpropyleneglycol. In the emulsion system, the size of ovolecithin liposome was increased and the clearness of the phospholipid bilayer was reduced as increasing the diethylether concentration. In the twophase solvent system, the rapidest reaction was obtained when water-organic solvent ratio was close to 1. The ratio of aqueous phase. however, should be lowered to 37% to gain the sole product of transphosphatidy1ation, without phosphatidohydrolysis.

The insecticidal protein gene in the pKL-20-1 clone derived from Bacillus thuringiensis serovar. kurstaki plasmid was subcloned in the plant shuttle vector, pGA643. The 7.3 kb fragment was cloned in the BglII and Hpal sites of pGA643 vector and expressed in E. coli S17-1, which produced insecticidal proteins killing Bombyx mori larvae. The clone was named pHL-20. The protoplast formation, calli induction and plantlet regeneration of Nicotiana tabacum was carried out. A tremendous number of mesophyll protoplasts of N. tabacum were formed, up to 7105 protoplast per ml, for 20 hours in darkness in the enzyme solution of 0.5% cellulase and 0.1% macerosin, pH 5.8. The viabilities of the protoplasts were maintained above 80% for 6 days in the media containing 2mg/1 of NAA and 1mg/1 of kinetin. Calli were induced from the protoplasts and leaves of the N. tabacum on MS medium containing 0.5mg/1 BAP. Under the culture conditions the protoplasts underwent repeated cell division into calli. Plantlets were regenerated from callus cultures derived from protoplast and leaves. Shoots were induced in a medium containing 1mg/1 of BAP.

In this paper the effect of light on non-aerated Baker's Yeast(Saccharomyces cereuisiae) production and the protein excretion to the extracellular fluid is studied. Previous results in our laboratory indicate that at pH=5 and T-32℃ yeast may be affected by light, but those differences seem to be within statistical variation of the data. In this paper, cell and extracellular protein concentrations along with redox potential are monitored for batch fermentations in the presence and absence of light at pH levels of 3 and 5 and at 31℃, in order to explore whether possible light effects can be more readily discerned at lower pH values. Yeast particle size distributions are also determined over the course of fermentation using a particle counter in order to add one more measuring tool to our usual cell and total protein measurements. An apparently noticeable difference in the redox potential is observed between the light and the dark runs for early times for the pH=3 runs. The particle size distributions show differences in the particle diameters between light and dark runs at pH=3, but those differences fall within one standard deviation of the mean particle diameters.

효소를 이용하여 대구피 젤라틴 가수분해물을 연속적으로 생산하기 위해 한외여과막 반응기에서의 젤라틴 가수분해 최적 공정조건 및 한외여과막 반응기를 장시간 작동하였을 때의 효소활성 저하원인을 규명하였다. 효소농도가 0.4mg/ml까지 증가함에 따라 기절의 전환율은 증가하였으나 기질의 경우는 농도가 증가함에 따라 전환율이 감소하였다. 유량(Flux)이 클수록 전환율은 감소되었으며 잔류시간이 길수록 전환율을 증가하였다. 한외여과막 반응기 장치의 작동시 가질의 전환율은 반응시간 1시간 부근에서 최대 전환율을 나타내었으며 시간당(8시간 작동) 평균 전환율 감소는 2.1%였다. 한외여과막을 통한 효소 누출은 반응시간 10~60분 범위에서 최대였으며 6시간 이후에는 거의 누출되지 않았다. 전체 효소량 중 누출량은 50℃에서 16%인데 비해 30℃에서는 12%였다. 그러나 효소 누출과 기질 전환율 사이에 명백한 상호관계는 나타나지 않았다. 막에 의한 효소의 활성저하는 작동시간 120분까지 나타났으며, 이때 초기속도의 36%가 저하하였다. 연속식(CSTMR)에서 Km 값은 회분식의 그것보다 20배 큰 반면 K2 10.5배 낮았다. 최적 가수분해조건은(기질농도 10%, , pH 8.0, 유량 7.31ml/min, 잔류시간 82분, S / E = 50w / w) 하에서의 생산량은 효소 mg당 가수분해물이 285mg으로 회분식 44.8mg에 비해 6배 이상이었다.

A continuous stirred tank membrane reactor(CSTMR ) was developed and optimized for the production of cod skin gelatin hydrolyzates using endo-protease Alcalase. A experimental design methodology was used to optimize the four performance variables: enzyme concentration, substrate concentration, permeate flux and reactor volume. All four variables studied had an effect on substrate conversion, with enzyme and substrate concentrations being predominant. Conversion increased with the increase in enzyme concentration, with the decrease in substrate concentration, at high volumes and low flux. A strong interaction was observed between enzyme and substrate concentrations and smaller interactions between enzyme and flux and substrate and flux. The optimum operating conditions for the CSTMR process for an initial substrate concentration for 10% were 50℃, pH 8, flux 7.3ml/min, residence time 82 min, and Alcalase to substrate ratio 0.02(w/w). A gradual decay in reactor activity during 8 hrs was 2.1% conversion/hr. Enzyme leakage through the 10, 000 MWCO membrane was 16% at 50℃ and 12% at 35℃, 6hrs. However, there was no apparent correlation between enayme leakage and substrate conversion. The Km value for the CSTMR was 20 times higher than the batch reactor. The productivity(expressed as mg product/mg enzyme) of the CSTMR was more than six fold higher than the batch at 50℃. The hydrolyzate was non-bitter.

본 연구에서는 매생물발효에 의한 L-phenylalanine생산에 미치는 배지조성의 영향을 살펴보았다. 탄소원의 선택은 L-phenylalanine생산에 큰 영향을 미침을 알았다. 자당, 포도당, 과당 혹은 그 혼합물을 탄소원으로 사용할때에 비해 당밀을 사용하는 것이 L-phenylalapine악 생산성을 크게 향상시켜 주었다. 최소배시의 사용시 미량의 염류들이 요구됨을 확인하였으나, 과량으로 첨가하면 더이상의 효고는 없었다. 또한 세포의 증식과 아미노산의 생산에 있어 적정량의 biotin과 thiamine이 필요함도 알I 수 있었다. Corynehacterium 균주외에 L.phenylalanine생산주로 알려진 조절기작을 상실한 영양요구성 변이주인 Brev-ihacterium lactofermenticum과 Arthrohacter citreus의 생산능을 조사해본 결과, Corynehacterium에 비해 생산성이 낮았으며 역시 영양요구 성질을 잃어버린 것으로 확인되었다.

In thisstudy, effects of medium components on microbial production of L-phenylalanine by Corynebacterium glutamicum were investigated. The effect of carbon source on the production of L-phenylalanine was significant. Molasses enhanced the production of L-phenylalanine compared to sucrose, glucose, fructose, or their mixture. It was noticed that trace salts were required for the cell growth and product formation in the minimal medium, but excess amounts of trace salts had no effect on the production of L-phenylalanine. It was also found that optimum amounts of biotin and thiamine were required for the cell growth and the production of L -phenylalanine.