• Journal of Microbiology and Biotechnology
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• 제18권8호
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• pp.1445-1452
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• 2008

An open reading frame (ORF) encoding a putative epoxide hydrolase (EHase) was identified by analyzing the genome sequence of Sphingophyxis alaskensis. The EHase gene (seh) was cloned and expressed in E. coli. To facilitate purification, the gene was fused in-frame to 6$\times$ histidine at the C-terminus. The recombinant EHase (rSEH) was highly soluble and could be purified to apparent homogeneity by one step of metal affinity chromatography. The purified SEH displayed hydrolyzing activities toward various epoxides such as styrene oxide, glycidyl phenyl ether, epoxyhexane, epoxybutane, epichlorohydrin, and epifluorohydrin. The optimum activity toward styrene oxide was observed at pH 6.5 and $35^{\circ}C$. The purified SEH showed a cold-adapted property, displaying more than 40% of activity at low temperature of $10^{\circ}C$ compared with the optimum activity. Despite the catalytic efficiency, the purified SEH did not hydrolyze various epoxides enantioselectively. $K_m$ and $k_{cat}$ of SEH toward (R)-styrene oxide were calculated as 4$\pm$0.3 mM and 7.42$s^{-1}$ respectively, whereas $K_m$ and $k_{cat}$ of SEH toward (S)-styrene oxide were 5.25$\pm$0.3 mM and 10.08$s^{-1}$ respectively.

• BMB Reports
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• 제29권2호
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• pp.111-115
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• 1996

A novel glutathione S-transferase from Pseudomonas sp. DJ77 was expressed in E. coli and purified by glutathione-affinity chromatography. The enzyme was composed of two identical subunits. The molecular size of the enzyme was 42 kDa by sephadex G-150 gel permeation chromatography and Mr of each subunit was 23 kDa by sodium dodecylsulfate-polyacrylamide gel electrophoresis. pI value of the enzyme was approximately 5.8 by isoelectric focusing. This enzyme showed the highest activity toward 1-chloro-2,4-dinitrobenzene as the electrophilic substrate. The relative activities toward p-nitrobenzyl chloride and 1,2-dichloro-4-nitrobenzene were 3.8% and 1.3% of the activity toward 1-chloro-2,4-dinitrobenzene, respectively. $K_m$ and $V_{max}$ values for 1-chloro-2,4-dinitrobenzene calculated by Lineweaver-Burk plot were 0.76 mM and $14.81\;{\mu}mol/min/mg$, respectively, and those for glutathione were 6.23 mM and $64.93\;{\mu}mol/min/mg$, respectively. The enzyme showed highest glutathione S-transferase activity at pH 8.0 and was stable between pH 6.0 and 9.0. The enzyme retained its activity up to $35^{\circ}C$ for 90 min but was unstable above $45^{\circ}C$.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 2002년도 창립10주년기념 및 국립독성연구원 의약품동등성평가부서 신설기념 국재학술대회:생물학적 동등성과 의약품 개발 전략을 위한 국제심포지움
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• pp.236-236
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• 2002

Pyruvate dehydrogenase phosphatase (PDP) is a mitochondrial protein serine/threonine phosphatase that catalyzes the dephosphorylation and concomitant reactivation of the pyruvate dehydrogenase componant of the pyruvate dehydrogenase complex (PDC). PDP consists of a Mg$\^$+2/ -dependent and Ca$\^$+2)-stimulated catalytic subunit (PDPc) of Mr 52,600 and a FAD-containing regulatory subunit (PDPr) of Mr 95.600. Catalytic subunit of pyruvate dehydrogenase phosphatase (PDPc) has been suggested to have three major functional domains such as dihydrolipoamide acetyltransferase(E$_2$)-binding domain, regulatory subunit of PDP(PDPr)-binding domain, and calcium-binding domain. In order to identify functional domains, recombinant catalytic subunit of pyruvate dehydrogenase phosphatase (rPDPc) was expressed in E. coli JM101 and purified to near homogeneity using the unique property of PDPc: PDPm binds to the inner lipoyl domain (L$_2$) of E$_2$ of pyruvate dehydrogenase complex (PDC) in the presence of Ca$\^$+2/, not under EGTA. PDPc was limited-proteolysed by trypsin, chymotrypsin, Arg-C, and elastase at pH7.0 and 30$^{\circ}C$ and N-terminal analysis of the fragment was done. Chymotrypsin, trypsin, and elastase made two major framents: N-terminal large fragment, approx. 50kD and C-terminal small fragment, approx. 0 kDa. Arg-C made three major fragments: N-terminal fragment, approx. 35 kD, and central fragment, approx. 15 kD, and C-terminal fragment, approx. 10 kD. This study strongly suggest that PDPc consists of three major functional domains. However, further study should be necessary to identify the functional role.

• Biotechnology and Bioprocess Engineering:BBE
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• 제10권2호
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• pp.128-135
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• 2005

Mixed-mode hydrophobic/ionic matrices exhibit a salt-tolerant property for adsorbing target protein from high-ionic strength feedstock, which allows the application of undiluted feedstock via an expanded bed process. In the present work, a new type of mixed-mode adsorbent designed for expanded bed adsorption, Fastline $PRO^{\circledR}$, was challenged for the capture of nattokinase from the high ionic fermentation broth of Bacillus subtilis. Two important factors, pH and ion concentration, were investigated with regard to the performance of nattokinase ad-sorption. Under initial fermentation broth conditions (pH 6.6 and conductivity of 10 mS/cm) the adsorption capacity of nattokinase with Fastline PRO was high, with a maximum capacity of 5,350 U/mL adsorbent. The elution behaviors were investigated using packed bed adsorption experiments, which demonstrated that the effective desorption of nattokinase could be achieved by effecting a pH of 9.5. The biomass pulse response experiments were carried out in order to evaluate the biomass/adsorbent interactions between Bacillus subtilis cells and Fastline PRO, and to demonstrate a stable expanded bed in the feedstock containing Bacillus subtilis cells. Finally, an EBA process, utilizing mixed-mode Fastline PRO adsorbent, was optimized to capture nattokinase directly from the fermentation broth. The purification factor reached 12.3, thereby demonstrating the advantages of the mixed-mode EBA in enzyme separation.

• 한국수정란이식학회지
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• 제24권4호
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• pp.259-263
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• 2009

Salivary lipocalin (SAL1) is a member of the lipocalin protein family that has a property to associate with many lipophilic molecules and was identified as pheromone-binding protein in pigs. Our previous study has shown that SAL1 is expressed in the uterine endometrium in a cell type- and implantation stage-specific manner and secreted into the uterine lumen in pigs. However, function of SAL1 in the uterus during pregnancy in pigs is still not known. To understand physiological function of SAL1 in the uterine endometrium during pregnancy in pigs, it needs to elucidate the ligand(s) for SAL1. Thus, to identify the ligand for SAL1 in the porcine uterus, we collected uterine luminal fluid from pigs on day 12 of pregnancy by flushing with PBS. Proteins from the uterine luminal fluid were separated by ion exchange chromatography and gel filtration. Fractions containing SAL1 protein were pooled and concentrated. Immunoblot analysis confirmed successful purification of SAL1. Then, we extracted lipids from the purified SAL1 protein and analyzed the lipids by liquid chromatography-mass spectrometry, and predicted to be steroid hormones and prostaglandins as SAL1 ligands. Results in this study showed that SAL1 protein in the uterine secretions has a small lipophilic molecule as a natural ligand. Further characterization of ligand extracted from purified SAL1 will be useful for understanding physiological function of SAL1 during pregnancy and its application to increase the pregnancy rate in pigs.

• 한국구조물진단유지관리공학회 논문집
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• 제14권3호
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• pp.152-159
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• 2010

최근 친환경 재료와 구조물에 대한 관심이 증대되면서 친환경 재료인 황토에 대한 연구가 활발히 진행되고 있다. 황토는 건설 및 마감 재료로 사용된 전통적인 친환경 건설 재료 중의 하나이다. 황토는 높은 축열성, 습도 조절 능력, 자정력, 탈취 및 항균성능, 인체에 유익한 다량의 원적외선이 방사되는 점 등 건설 재료로서 많은 장점을 갖고 있다. 그러나, 황토는 낮은 강도특성과 건조수축 균열 특성 때문에 현대 건설에 사용되지 않고 있는 실정이다. 최근 연구자들의 연구결과에 따르면, 황토는 플라이 애쉬나 포졸란 같은 천연 포졸란 재료로 사용될 수 있다. 본 논문에서는 황토와 슬래그, 재생 PET 섬유가 혼입된 콘크리트의 역학적 성질과 구조적인 휨 거동 실험을 수행하였다. 실험 결과 황토콘크리트의 압축강도와 탄성계수는 기준 시멘트 콘크리트 시편보다 상대적으로 낮게 평가되었으며, 철근 콘크리트 보의 휨 거동은 거의 유사하게 나타났다.

• Journal of Microbiology and Biotechnology
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• 제22권5호
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• pp.721-728
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• 2012

The human papillomavirus (HPV) is the main cause of cervical cancer in developing countries. Rapid diagnosis and initiation of treatment of the HPV infection are critical. Various methods have been employed to reduce the immunogenicity of antibodies targeting HPV serotypes. Nanobodies are the smallest fragments of naturally occurring single-domain antibodies with their antigen-binding site compromised into a single domain. Nanobodies have remarkable properties such as high stability, solubility, and high homology to the human VH3 domain. In this study, a phagemid library was employed to enrich for nanobodies against the L1 protein of the human papilloma virus. Binding reactivity of the selected clones was evaluated using phage enzyme-linked immunosorbent assay (phage-ELISA). Finally, two nanobodies (sm5 and sm8) with the best reactivity against the Gardasil vaccine and the purified HPV-16 L1 protein were expressed and purified using a $Ni^+$-NTA column. The accuracy of expression and purification of the nanobodies was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting assays. In vitro studies demonstrated that neutralization was achieved by the selected nanobodies. The ease of generation and unique features of these molecules make nanobodies promising molecules for the new generation of HPV diagnosis and therapy.

• 한국표면공학회지
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• 제50권2호
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• pp.98-107
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• 2017

Pd-based membranes have been widely used in hydrogen purification and separation due to their high hydrogen diffusivity and infinite selectivity. However, it has been difficult to fabricate thin and dense Pd-based membranes on a porous stainless steel(PSS) support. In case of a conventional PSS support having the large size of surface pores, it was required to use complex surface treatment and thick Pd coating more than $6{\mu}m$ on the PSS was required in order to form pore free surface. In this study, we could fabricate thin and dense Pd membrane with only $3{\mu}m$ Pd layer on a new PSS support manufactured by metal injection molding(MIM). The PSS support had low surface roughness and mean pore size of $5{\mu}m$. Pd membrane were prepared by advanced Pd sputter deposition on the modified PSS support using fine polishing and YSZ vacuum filling surface treatment. At temperature $400^{\circ}C$ and transmembrane pressure difference of 1 bar, hydrogen flux and selectivity of $H_2/N_2$ were $11.22ml\;cm^{-2}min^{-1}$ and infinity, respectively. Comparing with $6{\mu}m$ Pd membrane, $3{\mu}m$ Pd membrane showed 2.5 times higher hydrogen flux which could be due to the decreased Pd layer thickness from $6{\mu}m$ to $3{\mu}m$ and an increased porosity. It was also found that pressure exponent was changed from 0.5 on $6{\mu}m$ Pd membrane to 0.8 on $3{\mu}m$ Pd membrane.

• 한국식품영양학회지
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• 제22권2호
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• pp.261-271
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• 2009

Deep sea water exists at depths of over 200m under the sea. As no sunlight reaches it, photosynthesis does not take place within it, and it contains no organic matter. In addition, its temperature is maintained at a stable low level throughout the year, so it does not get mixed with the sea water on the surface. It contains a large amount of nutritious salts, whose cleanness is maintained. It is a marine resource that has matured for a long period of time. Research into deep sea water, which started in the 1970s, has been made around the whole world, including the USA and Japan. In Korea, research has been active in this area since 2000. As there has been a good amount of research into industrial applications for deep sea water, since 1993, patents for the relevant technologies have been applied. This paper intends to provide a resource to researchers of deep sea water, by summarizing of all domestic deep sea water-related patents applied with Korean Intellectual Property Office from 1993 to 2008. This research was conducted using a computer and KIPRIS Database owned by the Korea Institute of Patent Information. 'Deep sea water' was used as the search keyword. A total of 222 Korean patents relating to deep sea water have been registered on the basis of IPC. Of these, 126 patents relate to the manufacturing and the treatment of foods, foodstuffs, or non-alcoholic beverages(A23L), while 50 patents relate to the production for medical, dental, or cosmetic purposes(A61K). 38 patents relate to water purification, treatment of wastewater, sewage and sludge (C02F), while 8 patents relate to fishery and farming(A01K). In summary, it was found that studies for the practical use of deep sea water have been conducted in relation to the manufacturing and the treatment of foods, foodstuffs, beverages, and cosmetics.

• Journal of Microbiology and Biotechnology
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• 제14권6호
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• pp.1142-1149
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• 2004

Streptococcus mutans has the capacity of inducing dental caries. Thus, to develop a novel way of preventing dental caries, a cell wall hydrolase-producing strain was isolated and its characteristics were investigated. Among 200 alkalophilic strains isolated from soil, 8 strains exhibited lytic activities against Streptococcus mutans. However, strain YU5215 with the highest cell wall hydrolase activity was selected for further study. Strain YU5215 was identified as a novel strain of Bacillus based on analyzing its 16S rDNA sequence and Bergey's Manual of Systematic Bacteriology, and thus designated as Bacillus mutanolyticus YU5215. The optimal conditions for the production of the cell wall hydrolase from Bacillus mutanolyticus YU5215 consisted of glucose ($0.8\%$), yeast extract ($1.2\%$), polypeptone ($0.5\%$), $K_{2}HPO_{4}\;(0.1\%$), $MgSO_{4}{\cdot}7H_{2}O$ ($0.02\%$), and $Na_{2}CO_{3}\;(1.0\%$) at pH 10.0. Bacillus mutanolyticus YU5215 was cultured at 30^{circ}C for 72 h to produce the cell wall hydrolase, which was then purified by acetone precipitation and CM-agarose column chromatography. The molecular weight of the lytic enzyme was determined as 22,700 Da by SDS-PAGE. When the cell wall peptidoglycan of Streptococcus mutans was digested with the lytic enzyme, no increase in the reducing sugars was observed, while the free amino acids increased, indicating that the lytic enzyme had an endopeptidase-like property. The amino terminus of the cell wall peptidoglycan digested by the lytic enzyme was determined as a glutamic acid, while the lytic site of the lytic enzyme in the Streptococcus mutans peptidoglycan was identified as the peptide linkage of L-Ala and D-Glu.