• Journal of Microbiology and Biotechnology
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• 제6권1호
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• pp.1-6
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• 1996

A bacterial strain NS70 producing an alkaline protease was isolated from soil samples taken near a hot spring and identified as Bacillus licheniformis by its morphological and physiological properties and cellular fatty acid analysis. The isolated alkaline protease was purified by ammonium sulfate fractionation, DEAE-, CM-, and Phenyl-Sepharose column chromatography. The molecular weight of the purified enzyme was estimated to be 32, 000 Da by sodium dodecylsulfate polyacrylamide gel electrophoresis. Its optimal pH and temperature for proteolytic activity against Hammarsten casein were 12 and $65^{\circ}C$, respectively. The enzyme was stable at alkaline pH range from 6.0 to 12.0, and fairly stable up to $65^{\circ}C$. The enzyme was inhibited by phenylmethylsulfonyl fluoride but not by EDTA and N-ethylmaleimide indicating that the enzyme is serine protease. Enzyme activity was markedly inhibited by $Hg^{2+}$ and $Cu^{2+}$. Autolytic phenomena were observed on purified protease NS70 but autolysis was reduced by the addtion of $Ca^{2+}$ ion or bovine serum albumin.

• Journal of Microbiology and Biotechnology
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• 제3권1호
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• pp.31-38
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• 1993

A bacterial strain KY-l isolated from sewage was able to produce an extracellular agarase(agarose 4-glycanohydrolase. EC 3.2.1.81). The strain KY-1 was identified as Cytophaga fermentans subsp. agarovorans based on its morphological and physiological characteristics. The agarase was purified by ammonium sulfate precipitation followed by DEAE-Sephadex A-50. Bio-Gel P-100. and CM-Cellulose column chromatography. The molecular weight of the purified enzyme was 24 kDa by SDS-polyacrylamide gel electrophoresis. The optimum temperature and pH for the enzyme activity were 30^{circ}C and 7.5, respectively. The enzyme activity was significantly inhibited in the presence of 0.1 mM $HgCl_2$. whereas it was elevated 3 times by $MnSO_4$ at 1 mM concentration. The Km value and Vmax were 16.67 mg/ml and 3.77 unit/ml.min. The agarase gene was cloned into Escherichia coli MC1061 using the plasmid vector pBR322. A 1.4 Kb DNA fragment of PstI-digested chromosomal DNA of C. fermentans KY-l was inserted into the PstI site of pBR322. expressed in the E. coli. and up to 60% of the total enzyme was extracellularly secreted. Enzymatic properties of the extracellular agarases produced by both the transformant and the donor were very similar in terms of optimal pH and temperature.

• 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.

• Journal of Microbiology and Biotechnology
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• 제20권10호
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• pp.1378-1385
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• 2010

A novel agarolytic bacterium, KY-YJ-3, producing extracellular agarase, was isolated from the freshwater sediment of the Sincheon River in Daegu, Korea. On the basis of Gram-staining data, morphology, and phylogenetic analysis of the 16S rDNA sequence, the isolate was identified as Cellvibrio sp. By ammonium sulfate precipitation followed by Toyopearl QAE-550C, Toyopearl HW-55F, and MonoQ column chromatographies, the extracellular agarase in the culture fluid could be purified 120.2-fold with a yield of 8.1%. The specific activity of the purified agarase was 84.2 U/mg. The molecular mass of the purified agarase was 70 kDa as determined by dodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimal temperature and pH of the purified agarase were $35^{\circ}C$ and pH 7.0, respectively. The purified agarase failed to hydrolyze the other polysaccharide substrates, including carboxymethyl-cellulose, dextran, soluble starch, pectin, and polygalacturonic acid. Kinetic analysis of the agarose hydrolysis catalyzed by the purified agarase using thin-layer chromatography showed that the main products were neoagarobiose, neoagarotetraose, and neoagarohexaose. These results demonstrated that the newly isolated freshwater agarolytic bacterium KY-YJ-3 was a Cellvibrio sp., and could produce an extracellular ${\beta}$-agarase, which hydrolyzed agarose to yield neoagarobiose, neoagarotetraose, and neoagarohexaose as the main products.

• Journal of Microbiology and Biotechnology
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• 제24권1호
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• pp.19-25
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• 2014

An extracellular agarase was purified from Bacillus sp. BI-3, a thermophilic agar-degrading bacterium isolated from a hot spring in Indonesia. The purified agarase revealed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with an apparent molecular mass of 58 kDa. The optimum pH and temperature of the agarase were 6.4 and $70^{\circ}C$, respectively. The activity of the agarase was stable at high temperatures, and more than 50% activity was retained at $80^{\circ}C$ for 15 min. Furthermore, the enzyme was stable in the pH range of 5.8-8.0, and more than 60% of the residual activity was retained. Significant activation of the agarase was observed in the presence of $K^+$, $Na^+$, $Ca^{2+}$, $Mg^{2+}$, and $Sr^{2+}$; on the other hand, $Ba^{2+}$, $Zn^{2+}$, $Cu^{2+}$, $Mn^{2+}$, $Co^{2+}$, $Fe^{2+}$, and EDTA inhibited or inactivated the enzyme activity. The components of the hydrolytic product analyzed by thin-layer chromatography showed that the agarase mainly produced neoagarobiose. This study is the first to present evidence of agarolytic activity in aerobic thermophilic bacteria.

• Fisheries and Aquatic Sciences
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• 제8권4호
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• pp.213-219
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• 2005

Vitellogenin (VTG) was purified from the blood plasma of estradiol-17$\beta$ ($E_2$)-treated male marbled sole (Limanda yokohamae) using gel filtration and anion exchange chromatography. The purity of the marbled sole VTG (msVTG) was confirmed by polyacrylamide gel electrophoresis (SDS-PAGE) and N-terminal amino acid sequencing. The purified msVTG was used to produce monoclonal and polyclonal antibodies in mice and rabbits, respectively, and the specificity of the polyclonal antisera for msVTG was confirmed by Western blot analysis. The antibodies cross­reacted with a protein of molecular mass approximately 160 kDa in the plasma samples of mature female marbled sole. No cross-reactivity was observed with the plasma of male fish. A direct non-competitive sandwich enzyme-linked immunosorbent assay (ELISA) was developed using the monoclonal anti-msVTG and polyclonal anti-msVTG antibodies, with purified msVTG as the standard protein. The values of the intra- and inter-assay variations were within the ranges of $8.l-9.8\%$ and $8.5-12.2\%$, respectively. The sensitivity was about 0.3 ng/mL. Serial dilutions of plasma from mature female sole reacted with the msVTG-antibodies in the sandwich ELISA, whereas the plasma from male fish did not. The results indicate that the maturation status of female marbled sole can be identified using a sandwich ELISA for msVTG.

• Bulletin of the Korean Chemical Society
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• 제30권6호
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• pp.1360-1364
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• 2009

The bacterium Sphingomonas chungbukensis DJ77 produces the extracellular polysaccharide gellan in high yield. Gellan produced by this bacterium is widely used as a gelling agent, and the enzyme UDP-glucose pyrophosphorylase (UGP) is thought to play a key role in the gellan biosynthetic pathway. The UGP gene has been successfully cloned and over-expressed in E. coli. The expressed enzyme was purified with a molecular weight of approximately 32 kDa, as determined by a SDS-polyacrylamide gel, but the enzyme appears as ca. 63 kDa on a native gel, suggesting that the enzyme is present in a homodimer. Kinetic analysis of UDP-glucose for UGP indicates $K_m$ = 1.14 mM and $V_{max}$ = 10.09 mM/min/mg at pH 8.0, which was determined to be the optimal pH for UGP catalytic activity. Amino acid sequence alignment against other bacteria suggests that the UGP contains two conserved domains: An activator binding site and a glucose-1-phosphate binding site. Site-directed mutagenesis of Lys194, located within the glucose-1-phosphate binding site, indicates that substitution of the charge-reversible residue Asp for Lys194 dramatically impairs the UGP activity, supporting the hypothesis that Lys194 plays a critical role in the catalysis.

• 미생물학회지
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• 제33권1호
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• pp.7-14
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• 1997

성장인자가 포함되지 않은 무기염 액체배지(ISP-4)에서 Streptomyces coelicolor A3(2)의 포자가 발아할 때, 성장인자의 주기적인 요구성이(양 등,1993_ 단백질과 RNA 합성과 어떤 관계를 가지고 있는지를 규명하고자 하ㅣ였다. 발아는 10시간 정도 걸렸으며 이때 성장인자의 요구성이 2시간 주기로 반복되는 것을 재입증하였다. 포자의 크기는 시간에 따라 증가하였으나, 포자수는 표준 평판계수법에서 감소하였다. 포자 집단은 생리적으로 살아있거나, 휴면 중인 포자와 죽은 포자로 구분될 수 있었다. 이러한 발아과정에서 포자를 acridine orange(AO)와 iodonitrotetrazolium chloride(INT)로 염색하여 형광현미경으로 관찰하였을 때 RNA와 단백질도 일정한 주기를 갖고 합성되었으며, 이 주기성은 성장인자의 요구 주기와 거의 일치하였다. 이로써 발아 초기에 포자 집단은 성장인자 중에서 특히 단백질 합성과 관련된 물질인 아미노산이 주기적으로 요구되고 있음을 토론하였다.

• 한국환경과학회지
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• 제4권4호
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• pp.335-344
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• 1995

Spermine이 녹화중인 녹두자엽의 엽록소-단백질 복합체(CPs) 및 틸라코이드막 단백질의 변화에 미치는 효과를 조사하였다. 녹화가 진행됨에 따라 Cps형성이 촉진되었으며, 특히 광계의 엽록소-단백질(CP I)이 다량 추척되었다. 광수화 엽록소 단백질(LHCP)은 48시간의 초기 녹화과정에서 중요한 단백질로 나타났다.Spermine처리는 초기녹화과정에서 틸라코이드막의 CPs 축척에 효과적이었다. 색소체막 단백질은 녹화과정에서 많은 변화를 나타내었는데, 56kD단백질은 전 엽록체체서 다량 관찰되었꼬 24kD 단백질은 전 처리구에서 뚜렷한 증가를 보여주었다.Spermine처리에 의해 틸라코이드막 단백질 형성은 대조구에 비해 다소 증가되었다. 이러한 결과들로부터 spermine은 녹화과정에서 색소체의 발달과 색소체막의 안정화에 중요한 작용을 하는 것으로 생각된다.

• 한국환경과학회지
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• 제4권4호
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• pp.33-33
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• 1995

Spermine이 녹화중인 녹두자엽의 엽록소-단백질 복합체(CPs) 및 틸라코이드막 단백질의 변화에 미치는 효과를 조사하였다. 녹화가 진행됨에 따라 Cps형성이 촉진되었으며, 특히 광계의 엽록소-단백질(CP I)이 다량 추척되었다. 광수화 엽록소 단백질(LHCP)은 48시간의 초기 녹화과정에서 중요한 단백질로 나타났다.Spermine처리는 초기녹화과정에서 틸라코이드막의 CPs 축척에 효과적이었다. 색소체막 단백질은 녹화과정에서 많은 변화를 나타내었는데, 56kD단백질은 전 엽록체체서 다량 관찰되었꼬 24kD 단백질은 전 처리구에서 뚜렷한 증가를 보여주었다.Spermine처리에 의해 틸라코이드막 단백질 형성은 대조구에 비해 다소 증가되었다. 이러한 결과들로부터 spermine은 녹화과정에서 색소체의 발달과 색소체막의 안정화에 중요한 작용을 하는 것으로 생각된다.