Earticle

본 연구에서는 하수일차슬러지의 가용화 및 산발효를 위한 pilot scale 세정산발효공정의 운전 특성을 검토하였다. 하수일차슬러지의 가용화 및 산발효 특성을 평가하기 위해 SRT, 세정수 및 운전온도를 변화시켜 실험을 수행하였다. 세정산발효공정의 가용화율은 온도가 높을수록 증가하는 반면, 산생성율은 중온조건(35℃)이 고온조건(55℃)보다 높게 나타났다. VS 감량은 약 56%, 슬러지 부피감량은 약 93%까지 나타났다. 가용화측면에서 세정산발효공정의 최적운전조건은 pH 9,35℃ 및 SRT 5일이 적합 할 것으로 판단된다.

Pilot scale study was carried out to produce Volatile Fatty Acids with primary sludge from municipal wastewater treatment plant. An acid fermenter was operated at pH 9, 35℃, SRT of 3.5-4.25d, using a final effluent as elutriating water(Mode-Ⅰ)and pH 9, SRT 5d, temperature of 35℃(Mode-Ⅱ), 55℃(Mode-Ⅲ), using a primarily treated water as elutriating water. Although solubilization rate was enhanced with the increase of temperature, the VFAs production rate was decreased. The VS reduction was shown approximately 56%, and the sludge volume reduction was 93%. The optimal conditions for solubilization was obtained at pH 9, 35℃ and SRT of 5d.

In order to understand the machanism of action and regulation of ${\beta}$-adrenergic receptor in terms of molecular level, the purification of receptor protein has a fundamental importance. Moreover, species differences among avian, amphibian and mammalian ${\beta}$-adrenergic receptors make it more important to purify mammalian ${\beta}$-adrenergic receptor. Because ${\beta}$-adrenergic receptor is an integral membrane protein, it must be solubilized from the membrane for the purification. The purpose of the present study was to solubilize and characterize the mammalian $\beta$-adrenergic receptor from guinea pig lung in quantities by more efficient and practical method eventually to purify receptor. Guinea pig lung membrane preparation was solubilized by sequential treatment of buffers containing low and high concentration of digitonin which are 0.2 and 1.2% respectively. About 50% of the total receptor pool was released by this double extraction procedure. The $\beta$-adrenoceptors in the digitonin extract were identified using the ${\beta}$-adrenergic antagonist, (-)-[$^3H$]-dihydroalprenolol ([$^3H$]DHA). The solubilized receptor retained all of the essential characteristics of membrane-bound receptor, namely saturability; stereoselectivity; high affinity to ${\beta}$-adrenergic drugs. For the measurement of soluble receptor activity, Sephadex G-50 chromatography method has been widely used. Inspite of its accuracy and wide acceptance, this technique employed troublesome column work which required long time to assay the activity of receptor. We employed another methods to measure receptor activity. When using 0.5% polyethylenimine pretreated GF/B glass fiber filter, filtration technique could be used to measure soluble receptor activity. This technique enabled us to reduce the total amount of time to assay by a factor of 4 as well as to detect soluble receptor. In the present study, we could establish more efficient and practical solubilization method of mammalian $\beta$-adrenergic receptor. The rapidity and high yield of this solubilization scheme, together with the favorable recovery of the receptor activity, are significant steps toward the ultimate purification of the mammalian $\beta$-adrenergic receptor. The result of this study together with more convenient purification method could provide large amount of purified receptor with ease for various research purposes.

A total of 35 phosphate solubilizing bacterial strains were isolated from waste mushroom bed of Agaricus bisporus in Buyeo-Gun, Chungnam and screened for the production of indole acetic acid (IAA). The best IAA producing strain was identified as Pantoea rodasii using 16S rRNA analysis. In addition to the IAA production, this strain could act as an efficient phosphate solubilizer (1100 μg ml-1 after 5 days of incubation) also. The selected strain was cultured under different conditions in order to assess the optimum conditions for maximum IAA production. The nutrient broth (NB) medium was recorded as the best medium, where the maximum IAA production (229 μg ml-1) was recorded at the start of stationary phase (12 hours after inoculation) of the bacteria growth. The performance of the strain was found to be maximum at the temperature of 30oC followed by 25oC. IAA production was found to be increased with increasing tryptophan concentration (from 0.1 to 0.6%), however beyond this limit, a slight reduction in IAA production was observed. The strains’ ability to produce IAA was further confirmed by extraction of crude IAA and subsequent TLC analysis. A specific spot from the extracted IAA preparation was found corresponding with the standard spot of IAA with same Rf value. The results of HPLC analysis conducted in identifying and quantifying the IAA production more precisely, are in agreement with the results of the assessment done with colorimetric method. As revealed by the results of the pot experiment, the isolated strain could significantly enhance the growth (as measured by shoot and root growth) of mung bean plants compared to that of non-inoculated plants. Therefore it can be concluded that the present strain, Pantoea rodasii has great potential to be used as bio-inoculants.

A total of 35 phosphate solubilizing bacterial strains were isolated from waste bed of Agaricus bisporus in Buyeo-Gun, Chungchugnam-do province and screened for the production of indole acetic acid(IAA). The best IAA producing strain was identified as Pantoea rodasii using 16S rRNA analysis. In addition to the IAA production, this strain could act as an efficient phosphate solubilizer (1100 μg ml-1 after 5 days of incubation) also. The selected strain was cultured under different conditions in order to assess the optimum conditions for maximum IAA production. The nutrient broth (NB) medium was recorded as the best medium, where the maximum IAA production (229 μg ml-1) was recorded at the start of stationary phase (12 hours after inoculation) of the bacteria growth. The performance of the strain was found to be maximum at the temperature of 30°C followed by 25°C. IAA production was found to be increased with increasing tryptophan concentration (from 0.1 to 0.6%), however beyond this limit, a slight reduction in IAA production was observed. The strains’ ability to produce IAA was further confirmed by extraction of crude IAA and subsequent TLC analysis. A specific spot from the extracted IAA preparation was found corresponding with the standard spot of IAA with same Rf value. The results of HPLC analysis conducted in identifying and quantifying the IAA production more precisely, are in agreement with the results of the assessment done with colorimetric method. As revealed by the results of the pot experiment, the isolated strain could significantly enhance the growth (as measured by shoot and root growth) of mung bean plants compared to that of non-inoculated plants. Therefore it can be concluded that the present strain, Pantoea rodasii has great potential to be used as bio-inoculants.

This study investigated the synergistic effect of single inoculation and co-inoculation of phosphate-solubilizing bacteria (PSB) Pantoea rodasil LH-1and Burkholderia stabilis HS-7. Phosphate-solubilizing abilities of these strains were assessed by measuring phosphorus content in culture media that were singly inoculated or co-inoculated with these strains for 7 days. The co-inoculation of P. rodasil LH-1and B. stabilis HS-7 was found to release the highest content of soluble phosphorus (783.41 μg mL-1) into the medium, followed by single inoculation of B. stabilis HS-7B (743.90 μg mL-1) and P. rodasil LH-1 (736.59 μg mL-1). The highest pH reduction, organic acid production, and glucose consumption were also observed in the medium inoculated with both the strains, compared with that in the medium inoculated with the strain alone. Results of a plant growth promotion bioassay showed 7.7% and 15.5% higher leaf and root growth, respectively, in romaine lettuce coinoculated with P. rodasil LH-1and B. stabilis HS-7 than those inoculated with the strain alone. However, no significant difference was observed between single inoculation and co-inoculation of these strains with respect to phosphorus release and plant growth. Although the results of the present study did remarkdly not show the synergistic effect of phosphate solubilization by co-inoculation of the PSB strains examined, these results indicate that treatment with PSB exerts a beneficial effect on crop growth.

This study investigated the synergistic effect of single inoculation and co-inoculation of phosphate- solubilizing bacteria (PSB) Burkholderia metallica JH-7 and Burkholderia contaminans JH-15. Phosphate- solubilizing abilities of these strains were assessed by measuring phosphorus content in culture media that were singly inoculated or co- inoculated with these strains for 7 days. B. metallica JH-7 was found to release the highest content of soluble phosphorus (140.80 μg mL-1) into the medium, followed by single inoculation of B. contaminans JH-15 (135.95 μg mL-1) and co-inoculation of two strains (134.84 μg mL-1). The highest pH reduction, organic acid production, and glucose consumption were observed in the medium inoculated with B. metallica JH-7 alone compared with that in the medium co-inoculated with both the strains. Results of a plant growth promotion bioassay showed 17.4% and 7.48% higher leaf and root growth, respectively, in romaine lettuce inoculated with B. metallica JH-7 alone than in romaine lettuce inoculated with a control strain. However, no significant difference was observed between single inoculation and co-inoculation of these strains with respect to phosphorus release and plant growth. Although the results of the present study did not show the synergistic effect of phosphate solubilization by the PSB strains examined, these results indicate that treatment with PSB exerts a beneficial effect on crop growth.

본 연구는 하수처리장에서 발생되는 생슬러지와 잉여슬러지를 대상으로 열적가용화 공정의 적용성을 평가하였다. 열적가용화 효율 및 특성평가는 각 슬러지를 대상으로 100 ~ 220oC 온도범위에서 30분간 전처리를 수행한 후 실시되었다. 그 결과, 가용화 온도가 상승함에 따라 SCODCr, NH4 +,VFAs 농도가 증가하는 경향을 확인할 수 있었다. 또한 COD 가용화효율 평가 결과, 온도 상승에비례하는 경향이 나타나 열적가용화에 의해 가수분해 및 산발표 단계를 촉진시킨 것으로 판단된다. BMP (Biochemical Methane Potential) 실험을 통해 생슬러지의 경우 220oC에서 가장 높은 biogas 생산량을 보여주었으나, 증가율은 5.6%로 열적가용화에 의한효과가 미미한 것으로 나타났다. 반면 잉여슬러지의 경우 최대 38.8% (180oC) 증가하여, 열적가용화 공정은 잉여슬러지에 대한 적용성이 더욱 우수한 것으로 나타났다.

In this study, an applicability of thermal pretreatment for primary and secondary sludge, which are generated in a sewage treatment plant, was evaluated. The efficiency and charateristics was investigated with each sludge after pretreatment under the condition of 100~220oC for 30 minutes. As the result, it was found that SCODCr, NH4 +, VFAs concentrations increased as the pre-treatment temperature increased. For COD solubilization, it was also highly dependent on an increase of temperature resulting in acceleration on hydrolysis and acid fermentation. In the BMP (Biochemical Methane Potential) experiment, for the primary sludge, it showed the higher biogas production rate at a temperature of 220oC, however, the effect was insignificant (5.6%). Whereas, for the secondary sludge, the increase on biogass production rate was 38.8% (180oC) and this means that the secondary sludge is more suitable for an applicability of thermal pretreatment.

슬러지 열가용화 시험을 위하여 파일럿 장치를 사용하였다. 슬러지의 습식산화 조건(250℃ 이상, 40 atm, 산화제 주입)에 필요한 높은 에너지를 사용하지 않고, 열가용화 조건(175~200℃, 18~20 atm, 산화제 주입 없음)을 이용하여 200℃, 20 atm, RT=60 min 에서 최적의 운전조건을 나타냈으며, 가용화에 의한 슬러지 내 미생물의 세포벽을 파괴함으로써 전체 SS제거율 60%를 달성하였다. 또한 가용화된 고분자 물질을 혐기소화에 유용하게 이용할 수 있도록 저분자 물질로 전환시켜 전환율(TVFA/SCOD)을 평가한 결과 20% 이상의 결과를 얻었다. 슬러지의 열가용화 전처리를 통하여 혐기소화조에서 OLR(Organic Loading Rate)를 향상시킬수 있었고, 높아진 혐기소화율을 기초로 가스 발생량도 월등한 슬러지 열가용화 시스템의 구축이 가능하였다.

Pilot scale system was used to test sludge thermo-solubilization. Without using energy for wet oxidation condition (250℃, 40 atm, oxidant addition), optimized operating conditions were attained at 200℃, 20 atm, RT=60 min and total SS removal efficiency of 60% due to the destruction of microbial cell wall in the sludge was achieved under thermosolubilization condition (175~200℃, 18~20 atm, no addition of oxidant). More than 20% of molecular weight conversion rate (TVFA/SCOD) was obtained for anaerobic digestion through transition of a higher molecular matter into lower one. Sludge thermo-solubilization for anaerobic digestion pretreatment enabled the improvement of OLR (organic loading rate) and the establishment of higher biogas production system based on the increased anaerobic digestion rate.

Olfactory receptors, G protein-coupled receptor (GPCR), are integral membrane proteins composed of seven transmembrane spanning domains. These are not easy to be overexpressed, solubilized and purified because of their structures and strong hydrophobicity. Purification of olfactory receptor, where only a few studies have been done, is critical step for developing protein-based olfactory biosensor. In this work, human olfactory receptor, OR2AG1, was overexpressed in Escherichia coli. Electrophoresis of the overexpressed protein, visualized with Coomassie Blue staining and by immunoblotting with glutathione S-transferase (GST) antibody, revealed bands of 53, 118 and 160KDa, which were identified as the monomeric, dimeric and trimeric forms of the receptor proteins, respectively. The protein was difficult to solubilize with many detergents and only N-lauroyl sarcosine was found to be suitable for efficient solubilization. Human olfactory receptor, OR2AG1, was purified and solubilized by N-Lauroyl Sarcosine after treating membrane fraction with Triton X-100. Purified protein can be used as the primary transducer of olfactory biosensor after reconstitution in phospholipid vesicles.

Apolipoprotein A-1 (apoA-I) forms discoidal membrane structure with phospholipids during the formation of high density lipoprotein (HDL) and plays a role in reverse cholesterol transport pathway. The discoidal membrane nanostructure, so called Nanodisc, can be easily assembled in vitro using apoA-I and phospholipids. As well as this easy assembly process, the small size, Sushi-like structure, particle size uniformity, and robustness in solution of the Nanodisc gives us a sense of various ways of biotechnological applications, which might be drug delivery system, membrane protein host or therapeutics for cardiovascular diseases. Coenzyme Q10 (CoQ10) is a fat-soluble, vitamin-like substance found in the cells of many organisms. It is used as a nutritional supplement and is beneficial in the treatment of human conditions. [1; 2]. The use of CoQ10 is also gaining popularity in the cosmetic industry owing to its antioxidant properties.In spite of the versatility of CoQ10, the molecule's long side chain of 10 isoprenoid units make CoQ10 poorly water-soluble, limiting its bioavailability and more various applications. In the present study, a novel CoQ10 nanoparticle was assembled by encircling isoprenyl chains with human apolipoprotein A-I protein (apoA-I). This nanostructure of CoQ10 is quite reminiscent of nascent high density lipoprotein (HDL) particle composed of phospholipids and cholesterol.

It is well-known that certain mixtures of surfactants can provide better performance than pure surfactants for a wide variety of applications. A stable water-in-oil(W/O) nano-emulsions was reported to be prepared using sorbitan monooleate (Span80), polyoxyethylene 20 sorbitan monooleate (Tween80), decane oil and water1). The w/o emulsion was prepared by spontaneous emulsification2). The organic phase was used to mix the n-decane and varying ratio (v/v) between Span80 and Tween80 surfactants. In this work, we investigated that a partial physical property of the emulsion such as partial molar volume of water, emulsion stability by size distribution along with time. Solubilization of fibroin and its hydrolysate was performed by using the emulsions of varying water fraction. Excess peptide precipitates were removed by filtration. The level of solubilization of peptide was between 0.1 - 1.1 mg/ml of transparent emulsion. Fibroin was solubilized upto 16 mg/L of transparent emulsion.

G-protein coupled receptors (GPCRs), a family of integral membrane proteins with seven transmembrane helices, are difficult to be overexpressed, solubilized and purified, especially from bacterial cell, because of their complicated structures and strong hydrophobicity. Before applying to chromatographic purification method, solubilization of GPCR expressed as inclusion body is critical step for GPCR production. In this work, a human olfactory receptor, hOR17-40, a family of GPCR, was overexpressed at high-level in E. coli mainly as insoluble form of inclusion body. Expressed human olfactory receptor was difficult to be solublilized by many detergents and chaotropes and sarcosyl and SDS was found to be suitable for effiient solubilization. Sarcosyl showed better solubilization ability than SDS and was thought to be the most suitable detergent for solubilization. Solubilization of inclusion body was performed with different concentration of sarcosyl and solubilized protein quantities were analyzed with immunoblotting and total protein assay. 5% sarcosyl was determined as optimal concentration for inclusion body solublization of the human olfactory receptor. After solubilization of GPCR, further chromatographic purification method can be applied to produce GPCR that is for being used in the fields of drug discovery and development of biosensor.

Astaxanthin (3,3'-dihydroxy-β,β-carotene-4-4'-dione), a natural pigment of pink to red color, is widely distributed in nature particularly in the skin layer of salmonoids and the crust of shrimp, lobster, etc. Recently, it was produced from the yeast culture of Phaffia rhodozyma. Because of its high thermal stability and antioxidant functionality, its applications can be extended into food, cosmetics, and pharmaceutical ingredient beyond the traditional feed additive. Because of its very high lipophilicity, astaxanthin has been extracted traditionally by strong organic solvents such as chloroform, petroleum ether, acetone, etc. In this study, we developed a surfactant-based solubilization system for astaxanthin, and used it to extract astaxanthin from disrupted yeast cells. Among Tween 20, Triton X-100 and SDS, Tween 20 was identified as the most suitable surfactant in terms of extraction capacity and safety. The ethylene oxide group of Tween 20 was identified as the most significant factor to increase the HLB value that determined the extraction capacity. The effects of micelle formation condition, such as the molar ratio of astaxanthin and Tween 20, pH, and ionic strength were also investigated. pH and ionic strength showed no significant effects. The optimal molar ratio between astaxanthin and Tween 20 was 1：12. Antioxidant activity of astaxanthin was higher than β-carotene and α-tocopherol. Astaxanthin in the crude extract from the yeast cell was more resistant to air and/or light degradation than pure astaxanthin, probably because of the presence of other carotenoids and lipids.

상 접촉법을 이용하여 BSA를 음이온 계면활성제인 AOT와 isooctane으로 구성된 역미셀상으로 가용화하였다. 본 연구에서는 pH와 염의 종류 및 농도가 가용화 효율에 미치는 영향에 주목하였다. 0.1 M의 KCl, NaCl, MgCl2 그리고 CaCl2와 같은 1:1 염 혹은 1:2 염을 수용액에 첨가하면 첨가한 염의 종류에 따라 pH=5～7사이에서 최대의 가용화 효율을 보였다. CaCl2와 NaCl의 경우, 1 M까지는 첨가한 염의 농도가 증가함에 따라 효율도 증가하였으나 MgCl2의 경우에는 특이한 경향을 보였다. 염의 농도가 낮거나 pH가 낮은 경우에는 단백질이 광범위하게 수용액상과 유기상의 계면에 침전되는 것은 주목할 부분이다. 역미셀의 water-pool 크기는 물에 대한 계면활성제의 몰비인 W0를 측정하여 추산하였다. MgCl2의 경우에는 pH변화에 관계없이 W0=20의 거의 일정한 값을 보인 반면 CaCl2와 KCl의 경우 염 농도가 증가함에 따라 W0는 감소하였다.

Bovine serum albumin(BSA) was solubilized into the reverse micellar phase consisting of sodium bis(2-ethylhexyl) sulfosuccinate (AOT) and isooctane using the phase transfer method. Of particular interest in this study were the effects of pH and the added salt type and concentration on the solubilization efficiency. When univalent or divalent salts such as KCl, NaCl, MgCl2, or CaCl2 were added to the aqueous phase at a concentration of 0.1 M, maximum solubilization efficiency was attained at a pH ranging from 5 to 7, depending on the added salt type. Increased salt concentration up to 1 M resulted in an increased solubilization efficiency for CaCl2 and NaCl, while the addition of MgCl2 beyond 0.1 M showed an anomalous trend. Further, it was noteworthy that to a large extent the protein precipitated in the interface between the organic and aqueous phases at lower pHs and lower salt concentrations. The size of the reverse micelle water pool was estimated by measuring the molar ratio of the surfactant to the water, W0. Irrespective of pH in the aqueous phase, the resulting value of W0 was almost constant, eg., 20 for MgCl2. However, the value of W0 decreased with increased salt oncentration in the cases of KCl and CaCl2.

양이온 계변활성제의 역미셀을 이용하여 단백질을 선택적으로 분리할 수 있는 분리조건을 밝히기 위해서 분자량과 등전점이 다른 여러 단백질의 용해에 미치는 pH, 이온 및 강도, 계면활성제의 종류 등 system para-meter 틀에 대해 연구 검토하였다. 등전점이 낮은 pepSIn 은 pH 5-7 이상에서, 등전점이 높은 ribotluclease-a 나 lisozym은 pH 11-12 이상에서 용해되어 단백질이 계면 활성제의 (+)전하와 반대전하언 (-)를 띠는 등전점 이상의 pH영역에서 용해가 가능하였다. KCI을 이용하여 이온강도를 증가시킴에 따라 단백질의 용해 정도는 전반적으로 감소하였다. 친수성부분은 동일하나 소수성 무분이 다콘 양이온계면활성제 CTAB, DDAB, TOMAC 을 이용하여 역미셀제를 변화시켰을 때, 소수성 탄화수 소사슬이 3개 인 TOMAC(Wo=5) 이 탄화수소사슬이 1개인 CTAB(Wo=28)와 2개인 DDAB(Wo=7-12)에 비해 작은 역셀을 형성하여 낮은 용해 정도플 보였 다. 찬은 DDAB에 대해서도 유기용매의 종류에 따라 W Wo가 달랐는데 benzene(Wo=7)에 비하여 ttrachloro-ethylene (Wo= 12)이 보다 큰 역미젤을 형성하고 용해 능력도 높았다.

The solubilization of BSA, pepsin, trypsin and ribonuclease-a in cationic reverse micellar solutions has been investigated. For complete solubilization into reverse micellar solutions, pH values of several pH units above the isoelectric point of each protein were required. Solubilization was observed to decrease with increasing ionic strength of KCI. The size of empty micelles showed no significant change with increasing ionic strength. Trioctylmethyl ammonium chloride (TOMAC) in cyolohexane showed the lowest solubilization for the proteins. The system didodecyl dimethyl ammonium bromide (DDAB)-tetrachloroethylene was capable of solubilizing larger amounts of proteins than the system DDAB-benzene. Cetyl trimethyl ammonium bromide(CTAB)-hexanol-isooctane system showed lower solubilization than DDAB-tetrachloroethylene system, while it had higher Wo value.

실크의 피브로인 단백짐의 가수분해에 의한 실크 펠타이드의 제조를 위하여 설크의 가용화 조건이 분자량 분포에 미치는 영 향을 고찰하였다 염 화칼숨, ethylenediamine 및 황산에 의해 가용화된 실크의 평균 분자량은 각각 41600, 3308과 1268 dalton 으로서 염화칼숨을 제외한 나머지 두 가용화 방법에서 평균 분 자량이 수천 dalton인 실크 용액을 얻을 수 있었으며 분산계수 는 3-4의 범위뜰 보였다. 가용화 및 분말화한 후 6시간 동안 단백질 분해효소 처리에 의하여 600-1200 dalton의 중량평균 분 자량을 갖는 살크 접타이드틀 얻을 수 있었다. 24시간 염산에 의한 산분해 처리에 의해 얻은 실크 웹타이드의 평균 분자량은 145 dalton으로 80%의 높은 유리 아미노산 함량을 나타내었다. 여러가지 가용화 방법 및 가수분해 방법 중 낮은 농도의 황산용 액으로 우선 실크를 가용화한 후 중화 및 효소처리에 의해 웹타 이드로 가수분해하는 방법이 과도한 아미노산의 유리를 억제하 면서 분자량의 조절도 용이할 것으로 판단되었다.

The effects of fibron solubilization conditions on molecular weight distribution of enzymatically-hydrolyzed silk peptides were investigated. The weight-averaged molecular weights of silk proteins prepared by solubilization with calcium chloride, ethylenediamine and sulfuric acid were 41600, 3308, and 1268 dalton, respectively. Silk peptides in the average molecular weight range of 600-1200 dalton were obtained by protease treatment from solubilized silk fibroin. After the acid hydrolysis of silk protein using hydrochloric acid for 24 hr, silk protein was hydrolyzed to peptides whose average molecular weight and free amino acid content were 145 dalton and 80%, respectively. It was possible to control molecular weight distribution of silk peptides by the combination of solubilization and hydrolysis methods. Among the various treatment methods, acid solubilization followed by protease treatment had an advantage of molecular weight control for the peptide production.