• Bulletin of the Korean Chemical Society
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• 제28권6호
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• pp.929-935
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• 2007

The objective of this study was to develop a rapid, simple, and qualitative acetylcholinesterase (AChE)- detection kit, based on a modification of the Ellman and ELISA methods, for the detection of organophosphorus (OP) and carbamate (CB) pesticide. The developed kits were used to screen a large number of agricultural samples (spiked and real) for OP and CB pesticide residues. AChE was extracted from the heads of honeybees (Apis mellifera L.) using Triton X-100, and was purified through 3 steps: diethylaminoethylcellulose chromatography (DEAE), affinity chromatography and membrane filtering, and Mono-Q column chromatography. Epoxy-activated Sepharose 6B affinity chromatography was used for large-scale purification. The presence of OP and CB pesticide residues in agricultural samples was assayed on the basis of AchE inhibition value. The presence (6 bands) or absence of some colored bands on the test line indicated a negative or positive result, respectively. The limits of detection for measured organophosphorus (OP) and carbamates (CB) pesticide residues in standard pesticide solutions and fortified samples were ranged from 0.50 to 2.50 ppm and 0.50 to 4.75 ppm, respectively.

• 한국식품과학회지
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• 제24권2호
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• pp.187-192
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• 1992

일반계와 통일계 쌀 전분에 대한 구조적 성질을 조사하고 그 차이점을 비교하였다. 쌀 성분의 호화온도 범위는 일반계가 통일계보다 넓었고 쌀 전분의 최대 흡수파장은 일반계가 통일계 보다 높았다. 고유점도는 전분, 아밀로오스와 아밀로펙틴 모두 일반계가 통일계 보다 낮았다. ${\beta}$-아밀라아제 분해 한도는 전분에서는 차이가 없으나 아밀로오스와 아밀로펙틴은 일반계가 통일계 보다 약간 낮았다. 아밀로펙틴의 평균 사슬 길이와 바깥 사슬 길이는 일반계가 통일계 보다 짧았으나 안쪽 사슬 길이는 차이가 없었다. 열수 가용성 전분과 아밀로오스 함량은 일반계가 통일계 보다 높았다. 또한 열수 가용성 전분을 겔 크로마토그래피한 결과는 품종마다 독특한 용출모양을 보였다.

• Journal of Microbiology and Biotechnology
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• 제14권3호
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• pp.576-583
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• 2004

GERI-155 is a macrolide antibiotic containing two deoxyhexose molecules and shows antimicrobial activities against Gram-positive bacteria. Deoxysugar biosynthetic gene cluster of GERI-155 from Streptomyces sp. GERI-l55 genome was cloned. Four orfs were identified and a putative orf presumed to be the dTDP g]ucose-4,6-dehydratase gene was designated as gerE. GerE was expressed in E. coli by using a recombinant expression vector pHJ1. The expressed protein was purified from E. coli cell lysate by using ammonium sulfate fractionation, and DEAE-sepharose CL-6B and hydroxylapatite column chromatography. The molecular mass of the expressed protein correlated with the predicted mass that was deduced from the cloned gene sequence data. The recombinant protein was a homodimer with a subunit relative molecular weight of 39,000 Dalton. It was found to have dTDP-glucose 4,6-dehydratase activity and also found to be highly specific for dTDP-glucose as a substrate. The values of $K_{m} and V_{max}$ for dTDP-g]ucose were $32\mu$M and 335 nmol $min^{-1}$ (mg protein)^{-1}$, respectively. dTTP and dTDP were strong inhibitors of the protein. $NAD^+$, the coenzyme for dTDP-glucose 4,6-dehydratase, was tightly bound to the expressed protein.

• Journal of Microbiology and Biotechnology
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• 제12권2호
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• pp.189-195
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• 2002

Using Streptomyces sp. YU100 isolated from Korean soil, the fermentative production of phospholipase D was attempted along with its purification and characterization studies. When different carbon and nitrogen sources were supplemented in the culture medium, glucose and yeast extract were found to be the best. By varying the concentration of nutrients and calcium carbonate, the optimal culture medium was determined as 2.0% glucose, 1.5% yeast extract, 0.5% tryptone 0.3% calcium carbonate. During cultivation, the strain secreted most of the phospholipase D in the early stage of growth within 24 h. The phospholipase D produced in the culture broth exhibited hydrolytic activity as well as transphosphatidylation activity on lecithin (phosphatidylcholine). In particular, the culture broth showed 8.7 units/ml of hydrolytic activity when cultivated at $28^{\circ}C$ for 1.5 days. The phospholipase D was purified using 80% ammonium sulfate precipitation and DEAE-Sepharose CL-6B column chromatography, which produced a major band of 57 kDa on a 10% SDS-polyacrylamide gel with purity higher than 80%. The enzyme showed an optimal pH of 7 in hydrolytic reaction, and at pH 4 in a transphosphatidylation reaction. The enzyme activity increased until the reaction temperature was elevated to $60^{\circ}C$. The enzyme was relatively stable at high temperatures and neutral pH, but significantly unstable in the alkaline range. Among the detergents tested as emulsifiers of phospholipids, the highest enzyme activity was observed when 1.5% Triton X-100 was employed. However, no inhibitory effect by metal ions was detected. Under optimized reaction conditions, the purified enzyme not only completely decomposed PC to phosphatidic acid within 1 h, but also exhibited higher than 80% conversion rate of PC to PS by transphosphatidylation within 4 h.

• KSBB Journal
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• 제18권4호
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• pp.306-311
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• 2003

융합단백질의 절단을 위해 EK를 고정화하여 액상 절단반응과 같은 80％의 절단수율을 얻을 수 있었다. 그리고 니켈 친화칼럼을 이용하여 간단한 정제공정을 구축하였다. 공유결합한 EK의 경우 니켈친화 결합한 EK보다 높은 재접힘 수율을 나타내었고 풀림과 재접힘을 이용하여 효소의 초기 활성을 회복함에 따라서 반복사용을 통한 경제적인 절단공정을 구축할 수 있게 되었다. 그러나 고정화 과정에서 효소의 활성이 감소하는 문제점과 고정화 수율을 높이기 위한 연구가 필요하다.

• Journal of Microbiology and Biotechnology
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• 제7권2호
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• pp.107-113
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• 1997

A strain producing a high amount of chitinase was isolated from soil, identified as Pseudomonas sp., and tentatively named Pseudomonas sp. YHS-A2. An extracellular chitinase of Pseudomonas sp. YHS-A2 was purified according to the procedure of ammonium sulfate saturation, affinity adsorption, Sephadex G-100 gel filtration and Phenyl-sepharose CL-4B hydrophobic interaction column chromatography. The molecular weight of the purified enzyme was estimated to be 55 kDa on SDS-PAGE was confirmed by active staining. Optimal pH and temperature of the enzyme are pH 7.0 and $50^{\circ}C$, respectively, and the enzyme is stable between pH 5.0 and 8.0 and below $50^{\circ}C$. The main products of colloidal chitin by the chitinase were N-acetyl-D-glucosamine and N,N'-diacetylchitobiose both of which were detected by HPLC analysis. The enzyme is supposed to be a random-type endochitinase which can degrade any position of ${\beta}$-l,4-linkages of chitin and chitooligosaccharides. The chitinase inhibited the growth of some phytopathogenic fungi, Fusarium oxysporum, Botrytis cineria, and Mucor rouxii and these antifungal effects were thought to be due to the characteristics of endochitinase.

• 한국잠사곤충학회지
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• 제44권2호
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• pp.64-68
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• 2002

본 연구는 곤충 유전자를 이용한 항세균성 펩타이드 생산 및 농업용 소재로서의 응용에 관한 연구로서 항세균성 단백질 누에신 유전자를 베큘로 바이러스 발현계(BEVS)를 이용하여 곤충세포주에서 발현한 후 누에신 단백질의 농업용 소재로서의 가능성을 모색하기 위해 농작물을 가해하는 감자 고추의 무름병을 일으키는 Pectobacterium carotovorum subsp. carotovorum, 가지 및 고추의 풋마름병을 일으키는 Ralstonia solanacearum, 양송이 버섯의 세균성 갈색 무늬 병을 일으키는 Pseudomonas tolaasii 및 무와 배추의 검은썩음병을 일으키는 Xanthomonas campestris pv. campestris 에 대해서 항세균 활성을 관찰하였다. 그 결과 Pectobacterium carotovorum subsp. carotovorum, Ralstonia solanacearum 및 Pseudomonas tolaasii에 대해 높은 활성을 나타내었으며 Xanthomonas campestris pv. campestris에는 활성을 나타내지 않았다. Ion exchange 및 gel filtration chromatography 를 수행하여 약 20 kDa의 성숙 누에신 단백질을 순수 분리하여 pH 및 온도에 대한 안정성을 조사한 결과, pH 2~12 완충액에서 30분간 처리하였을 때에도 항세균 활성이 그대로 유지되었고 10$0^{\circ}C$에서 2시간 처리시에는 활성이 안정되었으며 4시간 처리시에도 80% 정도로 유지됨을 확인함으로써 누에신 단백질은 pH 및 온도에 대한 안정성이 있음을 확인할 수 있었다.

• Journal of Microbiology
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• 제33권3호
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• pp.244-251
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• 1995

An extracellular protease of Salmonella schottmulleri was purified from culture filtrate by using 0-75% ammonium sulfate precipitation, DEAE Sepharose Fast Flow ion exchange chromatography, Ultrogel HA chromatography and Sephacryl S-200 HR molecular sieve chromatography. To measure enzyme activity, synthetic dipeptide substrate (CBZ-arg-arg-AFC) with low molecular weight was employed as substrate. The molecular weight of the purified enzyme was approximately 80 kDa when determined by gel filtration on Sephacryl S-200 HR and 73 kDa when estimated by SDS-PAGE. The isoelectric point was 5.45. The activity of the purified enzyme was inhibited by metal chelating agesnts such as EDTA and 1.10-phenanthroline. The divalent cations, such as Ca$\^$2+/, Zn$\^$2+/, Fe$\^$2+/, Mg$\^$2+/ enhanced its activity. These results suggested that it was a metalloprotease. It had a narrow pH optimum of 6.5-7.5 with a maximum at pH 7.0 and a temperature optimum of 40.deg.C. It was stable at least for 1 week at 40.deg.C and maintained its activity for 24 hours at 50.deg.C, but it was rapidly inactivated at 65.deg.C. This protease was shown to be sensitive to sodium 50.deg.C, but it was rapidly inactivated at 65.deg.C. This protease was shown to be sensitive to sodium 50.deg.C, but it was rapidly inactivated at 65.deg.C. This protease was shown to be sensitive to sodium 50.deg.C, but it was rapidly inactivated at 65.deg.C. This protease was shown to be sensitive to sodium dodecyl sulfate (SDS) and was inactivated in a dose-dependent manner. However, it was resistant to Triton X-100 and the activity was enhanced to 32.3% with treatment of 0.025% Triton X-100.

• Preventive Nutrition and Food Science
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• 제1권1호
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• pp.87-92
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• 1996

This study describes a stable and simple method for the measurement of cholesteryl ester transfer protein(CETP) activities using reconstituted HDL and LDL as substrates. Apolipoproteins (apo) A-I and -B were purified from hog plasma by a new strategy without ultracentrifugation and delipidation. a simple two-step column chromatography was administered. In the first step of phenyl-sepharose CL-4B column chro-matography, hydrophobic plasma proteins were isolated. The most hydrophobic proteins bound to the column appeared to be A-I and apo-B. Contaminat proteins were efficiently eliminated from the sample by washing the column with 0.3M NaCI containing buffer after loading the plasma on the column. Two pure proteins showing each single band on SDS-PSGE of apo A-I and apo-B were individually obtained by a subsequent gel filtration column chromatography(Sephadex G-200). This two-step purification was simple and inexpensive compared to the ultracentrifugation and/or delipidation method that are most commonly used. Reconstituted hight-density lipoproteins(HDL) and low-density lipoproteins(LDL) were prepared using the purified apo A-I and-B, respectively. When these artificially prepared HDL and LDL were used in the assays for CETP as the cholesteryl ester(CE) donor and acceptor respectively, the specific transfer of CE increased up to two fold compared to that used the native HSL and LDL.

• Preventive Nutrition and Food Science
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• 제1권1호
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• pp.111-116
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• 1996

Polyphenol oxidase(PPO) isolated from the crude extract of ascidian, Halocynthia roretzi, showed higher affinity for catechol than tyrosine or DL-DOPA. Successful enzyme assay could be performed at $25^{\circ}C$, 10min. by mixing 0.2ml of crude enzyme extract with 2.8ml of 0.13M catechol in 0.1M sodium phosphate buffer(pH 6.4). The specific activity of PPO which had been purified with a combination of ammonium sulfate treatment, ion exchange chromatography on DEAE-cellulose, and gel filtration on Sepharose 6B was 13-fold disc gel electrophoresis. The activity of PPO was stable from pH 5.0 to 8.0 and showed the peak activity at pH 6.4 .The optimum reaction temperature for PPO oxidation on catechol was 35$^{\circ}C$ and those enzyme were heat stable up to 4$0^{\circ}C$. Molecular weigth of the enzyme was estimated about 170kDa. One molecule was found to be composed of gour subunits. Two of them had molecular weigh of 55kDa and the others 30kDa. The {TEX}$K_{m}${/TEX} values, {TEX}$V_{max}${/TEX} and catalytic efficiency({TEX}$V_{max}${/TEX}/{TEX}$K_{m}${/TEX}) for catechol were 0.12mM, 2.5mM/liter/min. and {TEX}$0.18min^{-1}${/TEX} respectively. The substrate affinity and electrophorectic pattern suggested that the enzyme of ascidian was considered to be not tyosine but catechol oxidase.