Earticle

Polystyrene계 음이온 교환수지인 Diaion PA 412에 Aureobasidium pullulans 기원의 fructosyltransferase를 crude enzyme 상태로 고정화하여 fructo-oligosaccharides의 생산을 검토하였다. 고정화 효소의 최적 반응 pH 및 온도는 각각 pH 5.0, 55℃. 이었고 고정화에 의해 열안정성이 크게 증가하였다. 고정화 효소에 의한 fructo-oligosaccharids 생성의 효소 반응 경향은 free cell 및 soluble enzyme과 거의 유사하여 최종 반응산물 중의 당 조성 이 동일하였다. 고정화 효소를 repeated-batch 방법으로 운전하여 fructo-oligosaccharides 생산공정에 적용해 본 결과 50℃에서 20일까지 안정성을 유지하였다.

A fructosyltransferase from Aureobasidium pullulans was immobilized onto a polystyrene-type anionic ion-exchange resin and the production of fructo-oligosaccharides was Investigated by the immobilized enzyme. The optimum pH and the temperature of immobilized enzyme were found to be pH 5.0, 55℃ respectively. The thermal stability of the enzyme was greatly enhanced after immobilization. The reaction profiles of the immobilized enzyme was almost identical to those of the free cells and the soluble enzyme. The immobilized enzymes were stable up to 20 cycles without loss of initial activity in a repeated-batch operation 50℃.

다공성 실리카표변에 폴리에틸렌이민과 글루타르 알데히드로 전화당 효소를 고정화시키고 자당과 에 탄올로부터 메틸 프룩토시드를 합성한 결과 다음과 같은 결론을 얻었다. 다공성 설리카를 폴리에틸렌이 민으로 표면처리하여 얻어진 고정화 담체는 그 다공 성 구조와 표면화학적 특성이 효소의 고정화에 적합 하여 전화당 효소에 대한 고정화 함량 120mg/g, 활 성도 lOOD/mg을 얻어 배당체의 합성반응에서 필요 로 하는 고정화 촉매를 제조할 수 었었으며 고정화 담체 표변에 형성된 폴리에틸렌이민 피막은 효소 부근에 친수성 분위기를 유지하여 높은 효소활성을 나 타내었고 에틸 프룩토시드가 효소에 접근하여 가수 분해되는 반응을 억제하여 메틸 프룩토시드의 수율 과 농도를 크게 증가시켰다. 자당과 메탄올 수용액 으로부터 메틸 프룩토시드를 합성한 결과, 자당 농도 O.291mol/l , 메탄올 농도 30%(v/v), 반응온도 25℃, pH 4.8, 효소활성도 2U/ml일 때 자당의 전화 율 91.2%, 에틸 프룩토사드 농도 27.0g/l , 그 평균 수율 55.9%을 얻었으며, 위의 연구결과로부터 고정화 효소에 의한 배당체의 합성반응이 연속생산공정 으로 연구개발 될 수 있을 것으로 기대된다.

Methyl fructoside was synthesized from sucrose and methanol using an immobilized invertase. The enzyme was covalently bound by glutaraldehyde on porous silica coated with polyethyleneimine to give loading capacity of 120mg of invertase per one gram of dry porous silica and effective activity of 100U per one milligram of bound invertase. Polyethyleneimine coating imparted a hydrophillic character, good activity retention and high loading capacity to the surface of porous silica as well as hydrophillic microenviroment in the vicinity of bound invertase. The immobilized enzyme was formed into an alginate-enclosed silica bead to have enough activity for methyl fructoside synthesis from aqueous methanol-sucrose solution. Using the alginate-enclosed biocatalyst the yield of methyl fructoside was obtained as high as 55.9% from aqueous 30% (v/v) methanol and 0.291mo1/l sucrose with 2U/ml activity at 25℃, pH 4.8.

스티렌/아크렬 라텍스 폴리머를 폴리에릴렌이민으 로 표면처 리하여 얻은 PEI-microspheres는 그 표면 화학적 특성이 효소의 고정화에 적합하여 배당체의 합성반응에 서 필요로 하는 alginate-enclosed micro­s spheres biocatalyst를 제조할 수 있었고 라텍스 폴리머 표변에 형성된 폴리에틸렌이민 피막은 효소 부근에 친수성 분위기를 유지하여 높은 효소활성을 나 타내었을 뿐만 아니라, 생성된 배당체가 효소에 접 근하여 가수분해되는 반응을 억제하여 배당체의 수 율과 농도를 크게 증가시 켰으므로 alginate-en closed microspheres에 의한 배당체의 합성반응이 연속생산공정으로 연구개발 될 수 있다.

Latex microspheres of styrene/acryl copolymer with acrylamide functional group were used for the stable covalent immobilization of an enzyme applicable for enzymatic synthesis of glycoside. The latex microspheres were coated with polyethyleneimine to establish structural and functional properties relevant to the covalent Immobilization with a high retention of activity. Polythyleneimine-coated microspheres satisfactorily immobilized the invertase for methyl fructoside synthesis, and model reaction were formed into alginate-enclosed microspheres biocatalyst. Using the alginate-enclosed microspheres biocatalyst, the yield of model glycoside was obtained as high as 52.2% at concentration of aqueous 30%(v/v) methanol and 0.291mo1/1 sucrose solution with 2U/ml of activity. The present study showed that the latex microspheres were successfully applied to enzymatic synthesis of glycoside.

고정화 효소를 이용하여 콜레스테롤을 신속, 정확 하게 측정할 수 있는 Flow injection analysis(FIA) 시스댐을 개발하였다. 콜레스테롤 산화효소와 콜레 스테롤 에스테르 가수분해효소를 cantrall어 pore g glass과 glutaraldehyde를 사용해 얻어진 고정화 효 소를, 유리관에 충진해 얻어진 컬럼은 약 3-5 I. U. 의 활성을 나타내였다. 백금을 이용해 구성된 전극 은 +600mV에서 효소반응 결과 생성된 과산화수소 에 대해 특이적으로 응답하였다. 구성되어진 FIA시 스템의 분석능을 향상시키기 위하여 시료량, 캐리어 용액의 유속과 조성 풍의 변화에 따른 응당올 조사 하였다. 최적화된 운영조건하에서 유리 콜레스테롤 과 콜레스테롤 에스테르 표준용액에 대한 응답은 각 각 60, 400mg/ml까지 선형성(r=0.994 빛 0.998) 을 나타내였다. 모든 시료의 분석은 정확, 정밀( <2. 5 5%)한 것으로 조사되었다. 시간당 23회의 분석이 가능하였고, 500회의 간혈분석 후에 응답감도가 약 반으로 감소하였다.

A flow injection analysis(FIA) system was developed for the determination of cholesterol using immobilized cholesterol oxidase and cholesterol ester hydrolase. The enzymes were immobilized on controlled pore galas(CPG) by the glutaraldehyde method. The glass colunms packed with immobilized enzymes were found to contain 3-5 I.U. for each enzyme. A hydrogen peroxide sensitive electrode was contructed and applied to the FIA system. The operational conditions for FIA response were investigated and optimized with variation of sampling volume, flow rate and composition of carrier solution. The FIA response were linear upto 60 and 400mg/m1 for free cholesterol and cholesterol ester, respectively. All samples were analyzed with a good precision (<2.5% CV) and accuracy. 23 samples were mea sured succesively within about an hour. Intermittent assays of more than 500 times caused 50% decrease in response sensitivity.

생물고분자 생산을 위하여 오니에서 slime을 생산하는 Zoogloea ramigera 115를 선택하였다. 추출한 생물고분자는 카드뮴, 아연 같은 금속에 대해서 높은 흡착성능을 나타냈다. 특히 발효조 broth는 높은 흡착성능을 나타냄으로써 부가적인 분리공정없이 생물흡착제로서 사용 가능할 것이다. 생물고분자는 calcium alginate bead 형태로 고정화되어 유가금속 회수를 위하여 충전층 반응기에 사용되었다. 생물흡착제는 구리, 카드뮴, 망간, 아연에 대하여 80% 또는 그이상의 높은 제거율을 나타냈으며 흡착-탈착-재흡착 실험에서 높은 안정성을 나타냈다. 고정화된 생물고분자 시스템은 이온교환수지와 같은 다른 중금속 제거 시스템과 경쟁력 및 잠재적인 산업상 응용 가능성을 보였다.

Zoogloea ramigera 115, well known type of bacteria to produce slime in sewage plants, was selected for biopolymer production. The extracted biopolymer showed high uptake capacity of metals such as cadmium and zinc. Especially the fermentor broth itself showed high adsorption of metal and could be used a biosorbent without an additional separation process. Biopolymer was immobilized into beads of calcium alginate and used in a packed bed reactor for the purpose of valued metals recovery. The biopolymer showed high removal efficiencies of 80% or greater for Cu, Cd, Mn and Zn, and high stability in sorption-desorption-resorption experiments. The immobilized biopolymer systems were found to be comparable to other metal removal systems such as ion exchange resins and to be of potential industrial application value.

본 연구에서는 sMMO를 갖는 메탄 자화균인 M. triclwsporium OB3b를 이용하여 메탄올 생산을 위한 기초실험을 수행하였다. 중요한 결과를 요약하면 다음과 같다(Table 2). 1. 세포 내 NADH의 재생을 위해 개미산을 첨가 할 때 whole-cell의 sMMO 활성은 pH 7.0 및 30℃ 에서 최대값을 보이며 propylene을 기질로 할 경우 약 130nmol/mg cell min 정도이다. 2. 인산은 MMO와 MDH 활성을 모두 저해하나 M MDH에 대한 저해 정도가 훨씬 크므로 메탄올 합성 에 사용이 가능하다. Noncompetitive mode를 가정 할 때 저해상수는 각각 185mM(MMO) 및 42mM ( (MDH)이었다. 3. 메탄올은 MMO 활성을 저해하며 noncompeti­t tive mode를 가정할 때 propylene기질의 경우 2 21mM 이었다. 4. 균체 내 sMMO 활성은 성장이 멈춰진 상태에 셔 비교적 때}른 속도로 감소하며 고농도 인산용액에 서 그 속도가 더 빨라진다. 5. 인산농도 91mM에서 메탄은 메탄올로 산화되 어 축적되며 4.5시간 동안 에탄올의 생성속도는 평 균 79nmol/mg min이었다.

As an effort to develop an alternative transportation fuel, the production of methanol from methane gas was studied using the resting cells of an obligatory methanotroph, Methylosinus trichosporium OB3b. The reaction was carried out in high concentration phosphate buffer solutions with the flask-grown cells containing the exclusively cytoplasmic methane monooxygenase (sMMO) activity. The methanol accumulation rate was observed to be 79nmo1/mg·min during the initial 4.5hr. Phosphate-dependent inhibition was found for both sMMO and methanol dehydrogenase (MDH) activities, and the inhibition constants were 185mM and 42mM, respectively. The inhibition mode was noncompetitive. Methanol was found to be very inhibitory to the sMMO activity and the inhibition constant (noncompetitive) was 21mM when propylene was used as substrate. The sMO activity in the resting cells was declined very fast and the rate became very high during the methanol production. These results indicate that the use of M. trichosporium OB3b as a biocatalyst for the methanol production is heavily dependent on the stable maintenance of the whole-cell SMO activity as well as the effective alleviation of product inhibition.

펄프 폐수처리를 위한 효과적인 생물학적 처리법 으로서 세 가지 고정화 방법과 자유균체를 이용한 방법을 사용하여 실험하였다. 균체로서는 Phaηe­r rochaete chrysosporium을 이용하였다. 고정화방법 중 Ca-alginate gel을 담체로 샤용한 방법이 가장 우수하였다. 이 방법은 안정성과 처리 효율변에서 좋은 결과를 보였으며, 약 400시간 동안 균체의 커다란 상태변화없이 반응시킬 수 있었다. 다른 고정화 방법들과 자유균체를 이용한 방법들 의 가장 큰 문제점은 생물 반응기 안에서의 공기 c channelling 현상에 의한 산소전달 저항과 균체끼리 의 clogging으로 인한 반응기 내부의 제어가 힘들게 된 것이었다.

Three immobilization techniques and free cell system were tested to determine the most effective technique for the treatment of pulp waste effluent. The tests were conducted using Phanerochaete chrysosporium as a biocatalyst in a process designed to treat pulp waste effluent. The results show that Ca-alginate gel was the best immobilization material. The chosen material improved the stability and increased the removal efficiency of the system. The experiment using the chosen material was mom- bored for 400 hours with no significant changes in the state of the fungus. Common problems with other immobilization materials and free cell system were oxygen transfer resistance caused by air channelling and clogging in the bioreactor.

부산물이 거의 생기지 않는 반응조건에서 inulin의 산 가수분해 반응에 미치는 초음파의 영향을 조사했다. 반응조건은 50~60℃와 염산 0.1~0.3%(w/w)의 상당히 완화된 범위였으며, 항온조 내 반응기 위치와 기계적인 교반에 대한 영향을 조사했다. 초음파를 사용한 경우와 하지 않은 경우 fructose생성속도를 온도와 산의 농도변화에 따라 비교했으며 두 경우 활성화에너지는 25kcal/mol로 같았다. 초음파에 의한 반응속도 증가는 평균 22%였다.

The effect of ultrasound on the acid hydrolysis of inulin was studied under significantly mild reaction conditions, at which sugar degradation products were not detected. Reaction conditions were i the range of 50~60℃ and 0.1~0.3%(w/w) of HCl concentrations. The effects of reactor position inside water bath and mechanical agitation under ultrasound were investigated. The production rates of fructose with/without ultrasound irradiation were compared. The activation energies for both control and ultrasound reaction were the same, i. e., 25kca1/mo1, and ultrasound enhancement was average 22%.

The biodegradability of some anionic surfactants were investigated using biological oxygen consumption measurement at different temperatures. As test surfactants, soap, alkyl sulfate (AS), alpha olefin sulfonate (AOS), alkyl polyoxyethylene sulfate (AES), linear alkylbezene sulfonate(LAS), microbial surfactants such as sophorose lipid (sopholipid) and spiculisporic acid (S-acid), were used. The test solution were incubated at 5℃, 18℃ and 32℃, respectively. The comparative rates of biodegradation were in accordance with the results obtained from the surface tension measurement and methylene blue method. The results of comparative blodegradabilities of the surfactants were as follows; soap, AS>AES>AOS>LAS at 18℃ and 32℃. However, at, 5℃the biodegradation rate of soap was better than other surfactants. Considering the actual environment of the river, it was concluded that the biological oxygen consumption rate method at lower temperature was more practical than the current method such as methylene blue assay with adapted shaking flask culture at 25℃

Crown ether류인 dibenzo-18-crown-건를 carn­e er로 이용하여 액체막을 통해서의 금속이온의 이동 량을 picric acid의 absorbance를 ultraviolet spec trometer로 측정하여 평가하였다. 그 결과 dibenzo 18-crown-6에 의한 금속이온의 이동은 2가 금속이 온이 l가 금속이온 보다도 더 컷다. 이것은 이 계에 서는 이들 금속이온의 크기보다도 이온의 전하와 수 화(hydration)의 영향을 받았다. 이것은 18-crown-6가 Ca2+이온과 같은 2가 이온의 수송에 적합하다 는 것을 의미한다. 또한 dibenzo-18-cro뻐-6에 의 한 이온의 수송은 이온의 선택성을 갖지 않고 이온을 포착할 수 있는 능력을 갖는 것이 좋은 것으로 확인되였다.

In transportation the amount of metal ion by crown ethers, dibenzo-18-crown-6 were investigated using ultraviolet spectrometer. Transported the amount of one valence metal ion as $K+andi+$ was not so much. On the other hand, two valence metal ion increased by dibenzo-18-crown-6, which means that the ionic charge and hydration of two valence metal ion affected the carrying ability of crown ethers. The carrying ability of dibenzo-18-crown-6 was, therefore, adequate for two valence metal ion as Ca2+ and Ba2+. It was also suggested that transport metal ion by crown ethers, which is related rather the catching ability than the selectivity of metalion.

토양으로부터 세포외로 lipoxygenase inhibitor를 생산하는 균주를 screemng하여 우수한 저해능을 가지는 곰팡이 1균주 (L242H)를 분리하였고, 분리한 균주 L242H로부터 lipogenase inhibitor 생산조건을 검토하였다. 분리한 균주의 배양학적. 형태학적 특성을 조사해 본 결과 Penicillium속으로 동정되었다. Penicillium L242H의 플라스크 배양에 의한 lipoxygenase inhibitor 최적배양조건은 3.0% glucose, 0.4 % ammonium sulfate, 0.1 % potassium p phosphate (dibasic), 초기 pH 7.0이었고, 500ml용 shake flask에 배지 100ml를 넣어 8일간 배양했을 때 lipoxygenase inhibitor가 최대로 생산되었다.

The microorganisms producing a lipoxygenase inhibitor were screened from a wide variety of sources. The isolated strain was assigned to genus Penicillium by its cultural and morphological characteristics. The proper medium for the production of lipoxygenase inhibitor was composed of glucose 3.0%, ammonium sulfate 0.4%, and potassium phosphate (dibasic) 0.1%. The cultivation for lipoxygenase inhibitor production was carried out in 500m1 Erlenmyer flasks containing 100m1 of the medium at 30℃ by cultivating reciprocally. The highest lipoxygenase inhibitor production was observed after 8 days of cultivation. The inhibitor was the low molecular weight substance and inhibited specifically soybean origin lipoxygenase.

초산이온이 대장균 성장을 저해하는 현상을 규명 하기 위해서 M9 배지, LB 배지를 사용해서 플라스 크, 발효조 배양을 수행하였다. 대장균의 초산 생성 은 높은 포도당 초기 농도, LB 배지와 같은 복합 배 지에서 활발하였고, 플라스크 배양과 같은 pH 조절 이 되지 않는 배양방법에셔는 초산의 생성에 따른 pH 감소에 따라 세포성장이 많이 저해되었다. 초산 을 외부에서 첨가하여 세포성장에 마치는 영향을 조 사하여 보았는데 LB 배지를 사용한 플라스크 배양 에서는 첨가된 초산의 농도가 2g/l 이상, M9 배지 를 사용한 플라스크 배양에서는 초산의 농도가 4g/l 이상, 그리고 M9 배지를 사용한 발효조 배양에 셔는 8g/l 이상에서 초산의 성장 저해효과가 나타 다. LB 한천 배지를 볕에 깐 LB 액체 배지의 플라스 크 배양을 수행하였다. LB 한천의 양이 증가함에 따라서 한천에서 포도당의 방출이 일어나 최종 대장 균 세포 O.D.가 증가하였다.

In order to elucidate the effect of acetate ion on the growth of Escherichia coli, flask and fermentor cultures were performed using M9 and LB media. Acetic acid was secreted at a higher rate under the conditions of high glucose concentration as well as of richer medium, i. e., LB broth. The pH in flask culture could not be controlled as i fermentor and pH decreased with the formation of acetic acid. The inhibition effect of acetic acid was pronounced at a lower pH, and the effective inhibitory concentrations of acetic acid were 2.0g/l for LB flask culture, 4.0g/l for M9 flask culture, and 8.0g/l for M9 fermentor culture. Dialysis flask culture was designed to slowly provide E coli cells with glucose. Solid LB agar was layered under LB liquid medium with the variation of agar concentration and solid volume, The increase in the solid portion in the total volume(agar＋liquid) resulted in the increase of the final cell concentration. This can be ascribed to the fact that the larger solid phase behaves like a larger reservoir for glucose and controls the growth of E. coli with a controlled rate.

Thalicrtrum rugosum 식물세포 현탁배양에 의한 berberine 생산에 미치는 여러 가지 종류의 elicitor들의 영향에 관해 연구 하였다. 효모 유래의 elicitor, 15가지의 서로 다른 종류의 무생물 종류 elicitor, 3가지 곰팡이로부터 얻은 elicitor 들을 처리하여 세포의 생장 및 berberine 생산에 미치는 영향을 비교 분석해 본 결과, 이들 elicitor들은 T. rugosum에 의한 berberine 생산을 크게 증대시키지 못함을 확인하였다

Effects of various elictitors were investigated to enhance the production of berberine in plant cell suspension cultures of Thalicrtrum rugosum. Treatments of yeast elicitor, 15 different types of abiotic elicitors, 16 kinds of fungal elicitors from three species of fungi were performed. Cell growth and berberine production were examined and compared for both normal and elicitor treated cultures. No distinguished increases in berberine yield by the addition of elicitors could be attained.

Two strains were isolated from the vinegar of Korean traditional fermented rice wine and the vine gar of fermented persimmon, respectively. These strains, designated as KM and BPV, were identified as the genus Acetobacter with respect to morphological, physiological, and biochemical characteristics. The Isolates oxidized ethanol to acetate and over-oxidized acetate or lactate to CO2 and H2O. They were positive in catalase test, while being negative in oxidase, gelatin liquefaction, VP test, H2O production and indole formation tests. No -pyrones ware produced from glucose and fructose. KM was tolerant of 11% ethanol while BPV was relatively sensitive to ethanol at a higher concentration than 5%. The guanine-plus-cytosine contents of the DNA of KM and BPV strains were 53.8 and 56.6 mol%, respectively. The cellular fatty acid compositions contained in these isolates were saturated straightchain C14:0 and C16:0,, and unsaturated straight-chain C18:1. Major ubiquinone system of KM was Q-9, but that of BPV was Q-10. In morphophysiological and biochemical aspects, KM strain was similar to Acetobacter pasteurianus. However, BPV strain was different from other Acetobacter type strains.

Absidia zychae NRIC 1199 produced two forms of carboxypeptidase(CPZ-1 and CPZ-2) which were distinguished in their isoelectric points but had almost identical properties(1). The amino acid sequences for the N-terminal of both enzymes were the same (Tyr-Thr-Ser-Pro-Lys-Leu-Xaa-Asp-Pro-Asp-Val) and any significant difference was not observed between amino acid compositions of the two enzymes. The ouchterlony double diffusion technique using antibody raised against the CPZ-2 protein demonstrated a good cross-reaction between CPZ-1 and CPZ-2 Genomic Southern analysis showed only one gene encoding CPZ in the genome of Absidia zychae. However, a significant difference between two enzymes was observed on peptide map using Staphylococcus aureus V8 protease, distinguishable only one band, indicating that multiple forms of CPZ are caused by post-translational modification, such as deamidation.

Aspγgillus niger에 의한 -lucosidase의 최적 생산조건으로는 0.8% CMC, 0.5% bef extract, 0.3% Ca(NO3)2,O.O3%K2HPO4,0.03%FeSO4,0.05%Li2SO4).2%tween80,trace solution lml, 초기 pH4.0 배양온도 30℃, 배양기간 5-6일이었고, 그 배양액 중의 최종 효소 활성은 8.5-9.8unit/ml였다.

This study was designed to reveal the conditions for B-glucosidase production from Aspergillus niger. The maximal enzyme production was obtained when the fungus was cultured at 30℃ for 5~6 days in the optimal medium containing 0.8% CMC, 0.5% beef extract, 0.3% Ca(NO3)2, 0.03% K2HPO4, 0.03% FeSO4, 0.05% Li2SO4, 0.2% tween 80, trace solution 1.0ml and initial pH 4.0, and then final enzyme activity under above conditions was 8.5-9.8 unit/ml culture filtrate.