Penicillium janthinellum의 사멸균체를 이용한 염료의 제거

이제혁,전억한 경희대학교 생명자원과학연구원 1997 硏究論文集 Vol.18 No.-

사멸균체를 사용한 염료의 흡착율은 배지환경의 pH 감소에 따라 증가하여 pH 2의 경우 염료 Appolocion H-E7B의 흡착ㄹ은 pH6에서 보다 약 60배의 초기 흡착율이 증가하였고, 온도는 높을수록 염료의 흡착율이 증가하여 실험 조건중 가장 높은 온도인 40℃에서 흡착ㄹ이 가장 양호하였다. 종래의 염색폐수 처리 방법으로 처리가 어려운 reactive염료의 흡착율이 우수하였고, 또한 혼합 염료도 흡착율이 양호하여 성상이 불균질한 염색 폐수의 처리에 적용 가능성이 긍정적이었다. 사멸균체에 detergent를 첨가할 경우 염료의 흡착이 저해되는데, 이것은 detergent가 균체의 염료의 결합 부위와 경쟁적으로 결합하여 흡착능을 저하시키는 것으로 사료되며, 각종 금속 ion의 존재시에도 사멸균체에 의한 염료의 흡착에는 거의 영향을 미치지 않아 각종 이온들이 복합적으로 함유된 염색폐수의 처리에도 Pen. janthinellum의 사멸균체가 적합한 것으로 사료된다. It is difficult to remove dyes which are major pollutants in the texile industries. The biosorption was carried out with dead biomass which were prepared by drying and grinding process of harvested microbial cells. The maximum biosorption was obtained with dead cells at pH 2.0 and 40 C. The initial biosorption rates were found to be 0.06, 0.086, 0.079(mg/gㆍmin) for each Apollocion Red H-E7B, Apollofix Red SF-3B, and Apollocion Red H-E3B, respectively. The effects of detergents and ions on the initial biosorption rates were also investigated to gain higher efficiency of dye removal since wastewater contains some ions and detergents. However, detergents added to the modified cells reduced dye biosorption rate and ions used also had no influence on the dye biosorption rates.

고정화 Photobacterium phosphoreum과 휘발성 독성 물질과의 반응성

이은수,전억한 경희대학교 생명자원과학연구원 1999 硏究論文集 Vol.20 No.-

휘발성 독성 물질 측정용 biosensor로서 P phosphoreum을 sodium alginate로 고정화하여 독성물질에 대한 반응성을 조사한 결과 각각의 독성물질마다 특이한 hnearity 관계식이 설정되는 농도 범위가 있었고 이를 이용하여 EC_50을 계산할 수 있었다 IC_50과 비교해보면 EC_50을 이용하는 것이 시간과 비용을 줄일 수 있고 낮은 농도의 독성 물질에 대해 더 민감한 반응을 나타내었다 특히, 휘발성이 강한 독성 물질 일수록 휘발성 방지를 위해서는 세포를 고정화하는 것이 더 효과적이었다 따라서 휘발성 독성물질에 대해서 free cell보다는 고정화한 세포가 더 민감하게 반응하였고 sodium alginate 고정화 세포보다는 strontium alginate로 고정화된 세포가 민감한 반응을 나타내었다 Immobilization method was investigated in order to maintain the stability of bioluminescence of Photobacterium for monitoring the volatile toxic substances. Sodium alginate was used as an immobilization matrix for P. phosphoreum. For the relationship between bioluminescene of immobilized P. phosphoreum and toxic substances, kinetic parameters of specific rate (μ), ratio of bioluminescence intensity (Io/I), and gamma (γ) value were estimated. The bioluminescence intensity was found proportional to the concentration of toxic substances and the free cells showed more sensitivity than immobilized cells when volatile substances were exposed to the cells. However, bioluminescence increased slightly with time because of volatility. Furthermore, P. phosphoreum immobilized on strontium alginate were rather favored in comparison with cells immobilized on sodium alginate for the response to substances used.

Inulinase와 투과성이 향상된 Zymomonas mobilis를 이용한 Jerusalem artichoke로 부터의 sorbitol 생산

김인철,전억한 경희대학교 부설 식량자원개발연구소 1991 硏究論文集 Vol.12 No.-

The use of Jerusalem artichoke containing p-1, 2-fructose oligomer in the production of sorbitol that is used as food additive and converted to L-sorbose have been studied. Coimmobilization method of inulinase and oxidoreductase was considered for the simultaneous hydrolyzation of inulin and conversion of glucose and fructose liberated from inulin to sorbitol. Both inulinase and oxidoreductase were immobilized in chitin(5%, w/v) and k-carrageenan(4%, w/v). The activity of oxidoreductase was specified by permeabilization of Zymomonas mobilis cell with 0.2% CTAB (cetyltrimethylammoniumbromide). The use of inulinase for hydrolyzation of inulin resulted in the achievement of each 36.65g/ℓ of glucose and 85.32g/ℓ of fructose which are valuable substrates for sorbitol production. Using these hydrolyzates, accumulation of 36.13g/ℓ of sorbitol occurred at 38 C and pH6.2 in the reactor. When permeabilized cell and inulinase were coimmobilized, sorbitol produced at 28.lg/ℓ. That is low compared with 36.13g/ℓ in separated reactor system.

Photobacterium phosphoreum을 고정화하기 위한 최적 Carboxymethylcellulose(CMC) 담체의 농도결정

이용제,전억한 경희대학교 생명자원과학연구원 1999 硏究論文集 Vol.20 No.-

CMC 담체는 점도가 큰 물질로 luminometer tube내의 사료에 유동을 최소화함으로써 산소의 공급을 일정하게 하여 bioluminecsence의 intensity를 일정하게 측정 할 수 있고, P phosphoreum을 고정하여 30분 후에 bioluminescence의 안정성을 가지므로 바로 측정할 수 있는 장점을 가지고 있다 독성에 민감하고 bioluminescence에 영향을 주지 않는 CMC의 농도(w/v)의 결정은 중요하며 1%(w/v) CMC 담체에 고정했을 때 bioluminescence의 유지가 가장 안정했으며, Cr 화합물에 대한 민감하게 반응하는 CMC 담체의 EC_50값ㅇ르 г값을 이용하여 산출했을 때 1% CMC 담체에 고정하였을 때 적은 독성 농도에서 민감한 bioluminescence의 감소를 볼 수 있었다. 그러므로 독성모니터용 P phosphoreun의 고정화 담체로 CMC의 1%(w/v)가 최적 농도이다. Bioluminescence of Photobacterium phosphoreum has been used for the detection of pollutants in the environment. Immobilization method was used to maintain the stability of bioluminescence of P. phosphoreum. Carboxymethylcellulose was investigated for immobilization of P. phosphoreum as a matrix without disturbance in bioluminescence emission. Maintenance of bioluminescence was determined from the P. phosphoreum immobilized on the various concentrations of carboxymethylcellulose. Relatively high bioluminescence intensity was observed with cells immobilized on 1%(w/v) carboxymethylcellulose. The effect of carboxymethylcellulose concentrations on the sensitivity of Cr-compounds including Na_(2)CrO_(4), K_(2)CrO_(4), CrO_(3), CrK(SO_(4))_(2) and CrCl_(3) to the bioluminescence intensity was investigated. The calculated EC_(50) showed that the linear relationship between such substances and bioluminescence.

자연분해성 Polymer blend와 이 blend의 생분해에 관한 연구

이제혁,이준열,전억한 경희대학교 환경연구소 1993 環境硏究 論文集 Vol.4 No.-

Several strains capable, of degrading PEGs(Polyethylene Glycols) were isolated and studied for their biodegradation ability of PEGs. We have isolated the strains that used the PEG-compounds as a sole carbon and energy source. We have screened the strains that reduced the concentration of PEGs in the liquid culture. The biodegradability was about 18.8% in PEG-8000 and 25.4% in PEG-10000. In order to produce PEG-film, EMAA/PEG and EAA/PEG blending ability was tested with 1R spectrum. Therefore, the blend was possible to be produced. EMAA/PEG and EAA/PEG blend film were produced and tested on the biodegradation ability. As a result that was tested with ASTM method, it was found that some of the blend films have the biodegradability. And we have tested identification of cells used in this experiment. Cells were identified as Pseudomonas species and Flavobacterium species.

Lactobacillus plantarum의 생육 억제에 미치는 요소 및 protoplast 형성에 관한 연구

박범식,--,전억한,조재선 경희대학교 부설 식량자원개발연구소 1993 硏究論文集 Vol.14 No.-

발효중 L.plantarum의 생육 영향 실험은 다양한 인자를 이용하여 결정하였는데 당 종류 및 함량에 따른 성장 효과는 glucose가 20%일때 발효성 당 기질의 활용율이 높았으며, pH 저하 속도 역시 가장 빨랐다. 초기 pH를 6.5에서 3.0까지 acids 첨가로 고정시켜서 초기 pH에 따른 발효중 pH의 변화 정도은 접종 초기 pH와 관계없이 4.0 이하에선 pH 저하가 거의 일어나지 않았으나 세포 농도는 증가하였다. 초기 pH 40 이상에서는 급격한 pH 저하가 있었다. 따라서 acidulants 첨가시 적어도 pH를 40이하로 낮추게 되면 세포 성장이 둔화 되어 김치 발효에 앞서 acididulation을 행할 수 없게 된다. 젖산균의 종류에 따라서 일정농도 이상의 염농도에서 생육이 중지되거나 삼투압이 세포내보다 클때에는 세포가 파열하여 생육할 수 없다. 염농도 1%에서 3%까지 증가함에 따라 세포상장에 어느정도 영향을 미쳤으나 pH저하에는 거의 영향을 미치지 않았다. DEDT와 Butylated hydroxyanisol은 생장 억제제로 각 1%, 0.04% 첨가한 경우 접종초기와 배양후의 pH 변화는 거의 또는 전혀 일어나지 않았으며, Zn을 첨가하였을때도 pH 변화가 발견되지 않았다. Protoplast를 생성하기 위하여 세포벽 용균 효소로 lysozyme 40㎍/ml과 protoplast 형성율이 가장 좋은 생육 시기인 15hr으로 결정하였다. 30℃, ph75에서 현저한 흡광도 감소 경향과 protoplast 형성율도 우수하게 나타났다. 15% sucrose, 20mM MgCl₂, 6mM CaCl₂에서는 흡광도 감소 경향을 보이지 않았으나, protoplast 형성율은 다른 농도에서보다 증가하는 경향을 나타냈다. Plasma expander로 알려진 gelatm을 첨가하였을 경우에는 6660㎚에서 유의적인 흡광도 감소경향을 보이지 않았으며 단지 protoplast를 형성시키는데 있어서 중요한 보조적 역할을 수행하는 것으로 사료된다. Lactic acid bacteria involved in KimChi fermentation was tested for disposition of preservation with initial pH and inhibitory activity against Lactobacillus plantarum. The inhibotory effects of various factors which are additional sugars, lowering initial pH, addition of salts on the cell growth were evaluated by measuring capacity of acid formation. The cell growth was found to be high at 20% glucose in accordance with fast falling of pH. It was revealed that the cell growth was inhibited with some decrease in pH when concentration of salt increased up to 3%. It was also found that Zn concentrations ranging from 0.01% to 0.04% did not show any influence on the cell growth. Microorganisms of L. plantarum were also used for the protoplast fusion to obtain the improved strains for KimChi fermentation. Protoplasts of L. plantarum was produced by lysozyme digestion at 30℃ for 180min. The strain was treated with 40㎍/㎖ of lysozyme in 30 mM Tris-Cl buffer(pH 7.5) containing 10% sucrose at the late logarithmic growth. It was found that the efficiency of protplast formation was high at 30℃, pH 7.5 determined by decrease in absorbance. Optimum concentrarions of sucrose, Ca^(2+), Mg^(2+) for protoplast formation were determined to be 15%, 20 mM and 6 mM, respectively.

염료제거능 곰팡이균주의 동정 및 염료 제거 특성에 관한 연구

이제혁,황규대,전억한 경희대학교 환경연구소 1995 環境硏究 論文集 Vol.6 No.-

F-2 isolates cultured on the agar plate containing the dyestuff has formed the clear zone around the strain. The morphology of F-2 was observed with microscope and identified as Penicillium janthinellum(subgenes Furcatum var. Divaricatum). The effects of pH on the absorbace of dyestuff almost not shown except above pH 12 and so the decrease of dye concentration during the incubation of Penjanthinellum is caused by the ability of Penjanthinellum that can remove the dyestuff. The pH and dry cell weight in the incubation of Penjanthinellum have changed after 18 hours and the decrease of the dye concentration in the medium has occured simultaneously. The rates of dye removal were good under the conditions of almost pH ranges except above pH 10 and 0.01g/100㎖ of dye was almost removed in over 3.0g/ℓ of starch concentration. In the case of l%(v/v) inoculum of Penjanthinellum in the 100㎖medium, 0.01g/100㎖ of dyestuff was almost removed in the 20 hours.

버섯의 갈변병 유발세균 P. tolaasii의 길항세균인 P. fluorescens의 분리 및 배지최적화

조남철,박범식,전억한 경희대학교 유전공학연구소 1991 遺傳工學論文集 Vol.3 No.-

버섯 갈변병 유발세균인 P. tolaasii에 길항성을 가진 P. fluorescens의 대량배양을 위하여 배지조성 및 배양의 최적조건을 확립하였다. 세포성장에 있어서 carbon 및 energy source인 glucose의 경우 30g/l일 때 세포농도가 가장 높았으며, yeast extract의 최적농도는 10g/l였다. 또한 균체의 구조합성에 있어 중요한 nitrogen source인 NH_(4)Cl과 (NH_(4))_(2)SO_(4)는 각각 1.0g/l와 0.1g/l일 때 세포성장이 가장 좋은 것으로 나타났고, sulfur source인 MgSO_(4)·7H_(2)O의 최적농도는 1.0g/l였다. 그리고 KH_(2)PO_(2)와 CaCl는 각각 1.0g/l와 0.1g/l일때 세포농도가 가장 좋았고, 온도 30℃, pH 6.0 그리고 초기 D.O가 68일 때 세포성장이 가장 높았으며, 균체 비증식속도(μ)와 생산성(P)도 높았다. P. fluorescens was studied in both batch and continuous culture in order to find out optimum conditions for cultivation. p. fluorescens is an aerobic bacteria and antagonistic to p. tolaasii which causes blotch disease on the mushroom cap. Cells of P. fluorescens were grown well on medium containing 30 g/ℓof glucose, whereas the growth was inhibited with the glucose concentration at higher than 30g/ℓ. The highest values of specific growth rate and productivities were obtained when using 10 g/ℓ of yease extract. Optimum concentrations of NH_(4)Cl and (NH_(4))_(2)SO_(4) for culture were found to be l.0g/ℓ and 0.1 g/ℓrespectively. The culture of MgS0_(4)·7H_(2)O was mostly about l.0g/ℓ as a sulfur source. It was also found that the cell concentrations were at the maximum level when grown on the medium containing l.0g/ℓ of KH_(2)PO_(4) and 0.lg/ℓ of CaCl_(2). Also, the optimum culture conditions were 30℃ and pH 6.0. Cultivation of P. fluorescens at high initial D.O value led to a decrease of bacterial productivity in batch culture. Maximum productivity was achieved at 68 for the initial D.O. value.

건조 담체 고정화 Zymomonas mobilis 세포의 oxidoreductase 활성도

장기효,이제혁,전억한 경희대학교 유전공학연구소 1991 遺傳工學論文集 Vol.3 No.-

박테리아인 Zymomonas mobilis는 glucose와 fructose를 각각 gluconic acid와 sorbitol으로 동시전환할 수 있다. Glucose fructose oxidoreductase에 의하여 glucose의 산화와 fructose의 환원이 일어난다. 균체를 cationic detergent인 CATB(cethyltrimethylammoniumbromide)로 처리하여서 투과성을 높인 후에 glutaraldehyde로 crosslinking 하였던 결과, 균체에서의 glucose-fructose oxidoreduc-tase의 유출이 감소되었으며 고정화균체의 안정성이 증가하였다. 투과성을 증가시킨 균체를 K-carra-geenan으로 고정화하여서 연속공정을 실시한 결과 효소의 안정성이 30일 이상 지속되었다. Kcarrageenan으로 고정화한 균체를 건조시 beads의 견고성과 저장성의 향상이 나타났다. 건조된 Kcarrageenan 담체의 V_(max)값은 39C와 pH6.2에서 free cells의 거의 절반값을 나타내었다. 연속공정에서 회석비율 0.1h^(-1)일때 wet beads와 건조 beads에서의 sorbitol 생산성은 각각 3.2∼3.5, 2.85∼2.9(g/l-h)를 나타내었다. The bacterium Zymumonas mobilis is able to convert glucose and fructose to gluconic acid and sorbitol simultaneously. The enzyme complex, which is responsible for glucose oxidation and fructose reduction, has been described as glucose-fructose oxidoreductase. After treatment of the cells with cationic detergent CTAB(Cetyltrimethylammoniumbromide) for permeabilization, the cells were treated with glutaraldehyde in order to crosslink enzymes. Glutaraldehyde treatment of the permeabilized cells could improve the stability of the system by decreasing the loss of glucose-fructose oxidoreductase. The methods of glutaraldehyde cross-linking of enzymes in CTAB treated cells immobilized in K-carrageenan showed a stable ability of enzymes in CTAB treated cells oxidoreductase for 30 days of process. K-carrageenan beads entrapped with permeabilized cells were dried to improve bead rigidity and storage stability. The value of V_(max) for the dry K-carrageenan beads was found to a 1/2 of that of free cells. It was shown that the productivities of the continuous process with wet K-carrageenan beads and dry beads at dilution rate 0.1h^(-1) were 3.2-3.5 g/l-h and 2.85-2.9 g/l-h, respectively.

독성 물질 측정을 위한 Photobacterium phosphoreum의 고정화에 관한 연구

이은수,이홍주,전억한 경희대학교 생명자원과학연구원 1998 硏究論文集 Vol.19 No.-

독성 물질 측정용 P phosphoreum의 고정화 물질 선택을위하여 고정화 방법을 크게 4가지로 나누어서 그 방법에 따라 각각 고정화 물질 한가지씩을 선택하여 P phosphoreum의 bioluminescence 안정성을 알아보았다. Polyacrylamide나 collagen에서는 bioluminescence가 유지를 못하고 20분 안에 급격히 떨어졌으나, alginate와 k-carrageenan은 온도를 높여야 gel이 형성되는 성질을 갖고 있기 때문에 저온성 발광 미생물인 P phosphoreum에 적합한 matrix가 되지 못한다. 따라서 P phosphoreum의 biluminescence를 안정되게 유지하면서 고정화가 용이한 polymer로는 alginate가 적합한 고정화 물질로 선정되었고 독성물질에 대한 P phosphoreum의 민감성을 bioluminescence의 변화로 살펴본 결과 각각의 독성 물질마다 특이한 linearity한 관계식이 설정되었고 EC_(50)를 쉽게 구할 수 있었다. The bacterial bioluminescence has been studied to detect toxic materials m foods and environment. A monitonng system can be developed with a fact that the bioluminescence is mhibited sensitively when the bioluminescent bacteria are exposed to toxic substances. However, a stability of bioluminescence must be improved for the purpose of developing continuous monitoring system. The mmobilization of Photobacterium phosphoreum, therefore, was investigated m view of long term of bioluminescence stability. Various matrixes, sodium algmate, k-carrageenan, collagen and polyacrylamide were studied for proper immobilization matnx of P. phosphoreum, in order to improve stability of bioluminescence. Collagen and polyacrylamide were shown to be madequate matenals for immobilization of P. phosphoreum since the bioluminescence decreased when cells were mixed with such matnx due to toxic effect of free radicals of matnx. In case of k-carrageenan, the biolumexence was stable compared with collagen and polyacrylamide. However, the k-carrageenan was not also a suitable matnx because cells were not able to mix with the matnx properly m low temperature at which gel is formed and, especially, cells of P. phosphoreum are psychrophilic luminescent bacteria. With a sodium algmate, the bioluminescence was stably maintained for 20 minutes and, in addition to that, a process of immobilization was simple for bioluminescent bacteria. Therefore, by using a sodium algmate as an mobduahon matrix, the relationship between biolummescence of immobilized P. phosphoreum and each toxic substance was shown linearity and could calculate EC_(50).