• Applied Microscopy
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• v.30 no.2
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• pp.185-191
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• 2000

An unusually large protein complex was found in the cytosol of the hyperthmophilic archaeon. Thermococcus profundus. The purified protein was shown to be a homomultimer of 93 kDa subunit (P93 complex). The complex is extremely heat stable. During 12 hrs incubation with SDS (final concentration 1%) at $85^{\circ}C$, no changed structure could be observed. Electron image analysis of negatively stained showed that the complex has a single, stable characteristic view and a well-preserved core with threefold rotational symmetry. The periphery of the assembly is composed of a nebulose, possibly flexible, component. Based on the projected structure suggest the P93 complex from T. profundus is composed 24 homomultimer.

• The Journal of Natural Sciences
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• v.14 no.2
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• pp.67-82
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• 2004

A thiol-specific antioxidant protein (TSA or TPx) was purified from Halophilic archeabacteria Halococcus agglomeratus, by DEAE-Cellulose, Phnyl, sepharose, Sephadex G-75, Sephacryl S-100, Sephacryl S-200, and Q-Wepharose FF. This protein exhibited the preventeive effect against the inactivation of glutamine synthehase (GS) activity was support by a thiol-reducing equicalent such as dithiothreitol. TPx activity was maximal at NaCl concentration above 500mM. The molecular mass of the protein was determinated to be 22-kDa by SDS-PAGE. The TPx purified from Halococcus agglomeratus seems to be similar to other TPx family, except for the salt requirement for the maximal antioxidant activity.

• Journal of Life Science
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• v.19 no.7
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• pp.994-1002
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• 2009

A bacteriocin-producing bacterium identified as Bacillus licheniformis was isolated from soybean sauce. Antibacterial activity was confirmed by paper disc diffusion method, using Micrococcus luteus as a test organism. The bacteriocin also showed antibacterial activities against Bacillus sphaericus, Lactobacillus bulgaricus, Lactobacillus planiarum, Paenibacillus polymyxa, and Pediococcus dextrinicus. Optimal culture conditions for the production of bacteriocin was attained by growing the cells in an MRS medium at a pH of 6.5~ 7.0 and a temperature of 37$^\circ$C for 36$\sim$48 hr. Solvents such as chloroform, ethanol, acetone, and acetonitrile had little effect on bacteriocin activity. However, about 50% of bacteriocin activity diminished with treatment of methanol and isopropanol at the final concentration of 50% at 25$^\circ$C for 1 hr. It was stable against a pH variation range from 3.0 and 7.0, but the activity reduced to 50% at a pH range from 9.0 to 11.0. It's activity was not affected by heat treatment at 100$^\circ$C for 30 min and 50% of activity was retained after heat treatment at 100$^\circ$C for 60 min, showing high thermostability. The bacteriocin was purified to a homogeneity through ammonium sulfate precipitation, SP-Sepharose ion-exchange chromatography, and reverse-phase high-performance liquid chromatography (HPLC). The entire purification protocol led to a 75-fold increase in specific activity and a 13.5% yield of bacteriocin activity. The molecular weight of purified bacteriocin was estimated to be about 2.5 kDa by tricine-SDS-PAGE.

• Parasites, Hosts and Diseases
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• v.26 no.2
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• pp.73-86
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• 1988

The sensitivity and specificity of crude and affinity-purified antigens of Clcnorchis sinensis obtained from the infected rabbits were studied. Stage-specific antigenic proteins from the eggs, metacercariae and adult worms were characterized by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and enzyme-linked immunosorbent astray (ELISA). The results were as follows: 1. The antibody.binding antigen (ABA) purified from whole worm crude antigen (IVWA) by CNBr-activated Sepharose 4B affinity chromatography made :l specific bands against rabbit antisera on Ouchterlony gel diffusion plate, while WWA made 7 bands. Major WWA protein bands by SDS-PAGE were found at 16, 300~18, 500 and 28, 000~29, 000 daltons, while major ABA protein bands were at 18, 000~21, 000 and 29, 000~31, 000 daltons. The reactivity of ABA with rabbit anti-sera in ELISA was remarkably less sensitive than that of WWA. 2. Molecular weights of egg antigen (EGA), metacercarial antigen (MEA) and adult worm antigen (WWA) of C. sinensis ranged from 15, 000-200, 000 daltons, 15, 000-100, 000 daltons and 11, 000~80, 000 daltons, respectively. Major WWA proteins consisted mainly of polypeptide bands of low molecular weight, less than 31, 000 daltons, while those of EGA and MEA consisted of higher molecular T.eights than 30, 000 daltons. 3. The ELISA reactivities of WWA to rabbit anti.sera were remarkably greater than those of MEA. EGA showed negative reaction throughout the experiments. WWA showed higher optical density (O.D.) than 1.0, when reacted with rabbit anti-sera obtained at 4~6 weeks after the infection. In the rabbit anti-sera later than 12 weeks after the infection, the O.D. reacting witll WWA showed a plateau without variation. MEA shoT.ed relatively low O.D. values (<0.6), when reacted with anti-sera from lightly in(ected groups throughout the experiments, althougll there were some wealth positive cases (O.D.>0.6) ill heavily infected groups. MEA reacted with rabbit anti-sera showed negative results on Ouchterlony gel diffusion plates. Summarizing the above results, it is suggested that the whole worm antigen prepared from the adult worms of C. sinensis is most highly antigenic. However, this antigen might reveal cross reactions with other trematodes such as Paragonimus westermani, therefore, purification of antigenic proteins from the crude antigen is essential 18 increase the sensitivity and specificity for the immuncdiagnosis of clonorchiasis.

• Korean Journal Plant Pathology
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• v.12 no.1
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• pp.99-108
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• 1996

3종의 빙핵세균 Peudomonas syringae 8401, Pseudomonas fuorescens 8701, Erwinia herbicola 8701의 세포 외막으로부터 아무런 변성제도 사용치 않고 sucrose density gradient centrifugation, Sephacryl gel filtration chromatography, DEAE-cellulose ion exchange chromatography, non-denaturing buffer를 이용한 PAGE, electroelution, SDS-PAGE를 통해 빙핵활성 단백질을 고도로 정제할 수 있었다. P. suringae와 P. fluorescens에서는 각각 3종류(155 kD, 75 kD, 50 kD)의 빙핵활성 단백질이, E. herbicola에서는 155 kD를 제외한 2종류(75 kD, 50 kD)의 빙핵활성 단백질은 이 연구를 통해 처음 밝혀진 것으로 , 지금까지 보고된 빙핵활성 단백질(150 KD 이상)보다는 훨씬 작은 것이다. 이는 빙핵활성을 나타내는 단백질의 기본단위는 이 실험의 결과만에 의하면 최대 50 kD임을 시사한다. 이들 단백질은 그 유래된 세균의 종류나 또는 단백질 분자량의 크기에 관계없이 모두 -5.5~7.5$^{\circ}C$에서 물을 동결시키는 높은 빙핵활성을 갖고 있었다. 이는 지금까지 보고된 어느 정제단백질의 빙핵활성보다 높은 것이다. 정제된 단백질의 빙핵활성은 trypsin 처리에 의해 상실되었고, pH6~8범위에서는 안정하였으며, pH5이하, pH9이상에서는 활성을 상실하였다. 보존온도에 대한 영향은 3$0^{\circ}C$이상이 되면 점차 활성이 감소하는 경향을 보이다 37$^{\circ}C$이상에서는 활성이 완전히 상실되었다. 금속이온으로서 Hg\ulcorner이온과 SDS에 의해 활성이 상실되었으나 phosphatidylinositol의 첨가에 의해서는 활성이 약간 증가(-1$^{\circ}C$)하였다.

• Journal of Soil and Groundwater Environment
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• v.9 no.4
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• pp.42-51
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• 2004

Peat humin(p-Humin), humic acid(p-HA) and fulvic acid(p-FA) were isolated from Canadian Sphagnum peat moss by dissolution in 0.1M NaOH followed by acid precipitation. After purification cycles, they are characterized for their elemental compositions and, acid/base properties. Functionalities and carbon structures of the humic fractions were also characterized using FT-IR and solid state $^{13}C$-NMR spectroscopy. Those results are compared with one another and with soil humic substances from literatures. Main purpose of this study was to present a chemical and spectroscopic characterization data of humic substance from peat moss needed to evaluate its environmental applicability. The relative proportions of the p-Humin, p-HA and p-FA in the peat moss was $76\%,\;18\%,\;and\;3\%$, respectively, based on the total organic matter content ($957{\pm}32\;g/kg$). Elemental composition of p-Humin were found to be $C_{1.00}H_{1.52}O_{0.79}N_{0.01}$ and had higher H/C and (N+O)/C ratio compared to those of p-HA($C_{1.00}H_{1.09}O_{0.51}N_{0.02}$) and p-FA($C_{1.00}H_{1.08}O_{0.65}N_{0.01}$). Based on the analysis of pH titration data, there are two different types of acidic functional groups in the peat moss and its humic fractions and their proton exchange capacities(PEC, meq/g) were in the order p-FA(4.91) >p-HA(4.09) >p-Humin(2.38). IR spectroscopic results showed that the functionalities of the peat moss humic molecules are similar to those of soil humic substances, and carboxylic acid(-COOH) is main function group providing metal binding sites for Cd(II) sorption. Spectral features obtained from $^{13}C$-NMR indicated that peat moss humic molecules have rather lower degree of humification, and that important structural differences exist between p-Humin and soluble humic fractions(p-HA and p-FA).

• Microbiology and Biotechnology Letters
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• v.44 no.2
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• pp.130-139
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• 2016

A bacterial strain, producing an excellent lipolytic enzyme, was isolated from the intestinal tracts of an earthworm (Eisenia fetida). The strain was identified as Aeromonas hydrophila by phenotypic, chemotaxonomic characteristics and 16S ribosomal DNA analysis, and was designated as Aeromona hydrophila PL43. The lipolytic enzyme from A. hydrophila PL43 was purified via 35−45% ammonium sulfate precipitation, DEAE-sepharose fast flow ion-exchange, and sephacryl S-300HR gel filtration chromatography. The yield of the purified enzyme was 3.7% and 2.5% of the total activity of crude extracts with p-nitrophenyl butyrate (pNPB) and p-nitrophenyl palmitate (pNPP) as substrates, respectively. The molecular weight of the purified enzyme was approximately 74 kDa using gel filtration, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and zymography. The optimal activity of purified enzyme was observed at 50℃ and pH 8.0 using pNPB, and 60℃ and pH 8.0 using pNPP. The purified enzyme was stable in the ranges 20− 60℃ and pH 7.0−10.0. The activity of purified enzyme was inhibited by PMSF, pepstatin A, Co²⁺, Cu²⁺, and Fe²⁺, but was recovered by metal chelating of EDTA. The K_m and V_max values of the purified enzyme were 1.07 mM and 7.27 mM/min using pNPB and 1.43 mM and 2.72 mM/min using pNPP, respectively.

• Korean Journal of Environmental Biology
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• v.35 no.4
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• pp.694-705
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• 2017

The aim of this study was to isolate and identify marine bacterium with anti-methicillin-resistant Staphylococcus aureus (MRSA) activity, and to purify the anti-MRSA compound, as well as to determine its activity and synergistic effects. Among the marine bacteria isolated in this study, the YJ-1 isolate had the strongest anti-MRSA activity. The YJ-1 isolate was identified on the basis of its biochemical characteristics and an analysis of 16S rRNA gene sequences. The YJ-1 isolate showed over 99.2% homology with Pseudomonas stutzeri, and was designated as a Pseudomonas sp. YJ-1. The optimal culture conditions were $25^{\circ}C$ and initial pH 7.0. For the purification of the anti-MRSA compounds, the YJ-1 was cultured in Pa PES-II medium, and the culture filtrates were extracted by ethyl acetate, hexane, and 80% MeOH. The 80% MeOH fraction was separated by a $C_{18}$ ODS column, silica gel chromatography and a reverse phase HPLC, to yield three anti-MRSA agents, the MR1, MR2, and MR3 compounds. When the MR1 compound of $250{\mu}g\;mL^{-1}$ concentration was applied to the MRSA cells, over 95% of bacterial cells was killed within 48 hr. Compared with vancomycin and ampicillin, the MR1 compound showed significant anti-MRSA activity. In addition, the anti-MRSA activity was increased by dose and time dependent manners. Furthermore, the combination of an MR1 compound with vancomycin produced a more rapid decrease in the MRSA cells than did the MR1 compound alone. Taken together, our results suggest that the Pseudomonas sp. YJ-1 and its anti-MRSA compounds could be employed as a natural antibacterial agent in MRSA infections.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.38 no.3
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• pp.148-152
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• 2005

A novel neuropeptide with a relaxing activity on the dorsal retractor muscle (DRM) was isolated from the whole body extract of the starfish, Asterina pectinifera. The peptide was purified by gel-filtration ion-exchange and $C_{18}$ reversed-phase HPLC. The complete amino acid sequence of this peptide, which was determined by automated Edman degradation and MALDI-TOF mass, was Phe-Gly-Lys-Gly-Gly-Ala-Tyr-Asp-Pro-Leu-Ser-Ala-Gly-Phe-Thr-Asp. A comparison of the amino acid sequence with those of other known neuropeptides revealed that the asteripectin was a novel neuropeptide with smooth muscle-relaxing activity on the starfish DRM. This peptide showed threshold response to relaxing activity on the DRM at $10^{-10}M$ and the maximal relaxing effect was $120{\pn}7.0\%$ at $10^{-5}M$. The relaxing activity of this peptide on the starfish DRM increased in a dose-dependent manner.

• Journal of Microbiology
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• v.45 no.2
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• pp.158-167
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• 2007

In this study, we have cloned a novel cDNA encoding for a papain-family cysteine protease from the Uni-ZAP XR cDNA library of the polychaete, Periserrula leucophryna. This gene was expressed in Escherichia coli using the T7 promoter system, and the protease was characterized after partial purification. First, the partial DNA fragment (498 bp) was amplified from the total RNA via RT-PCR using degenerated primers derived from the conserved region of cysteine protease. The full-length cDNA of cysteine protease (PLCP) was prepared via the screening of the Uni-ZAP XR cDNA library using the $^{32}P-labeled$ partial DNA fragment. As a result, the PLCP gene was determined to consist of a 2591 bp nucleotide sequence (CDS: 173-1024 bp) which encodes for a 283-amino acid polypeptide, which is itself composed of an 59-residue signal sequence, a 6-residue propeptide, a 218-residue mature protein, and a long 3'-noncoding region encompassing 1564 bp. The predicted molecular weights of the preproprotein and the mature protein were calculated as 31.8 kDa and 25 kDa, respectively. The results of sequence analysis and alignment revealed a significant degree of sequence similarity with other eukaryotic cysteine proteases, including the conserved catalytic triad of the $Cys^{90},\;His^{226},\;and\;Asn^{250}$ residues which characterize the C1 family of papain-like cysteine protease. The nucleotide and amino acid sequences of the novel gene were deposited into the GenBank database under the accession numbers, AY390282 and AAR27011, respectively. The results of Northern blot analysis revealed the 2.5 kb size of the transcript and ubiquitous expression throughout the entirety of the body, head, gut, and skin, which suggested that the PLCP may be grouped within the cathepsin F-like proteases. The region encoding for the mature form of the protease was then subcloned into the pT7-7 expression vector following PCR amplification using the designed primers, including the initiation and termination codons. The recombinant cysteine proteases were generated in a range of 6.3 % to 12.5 % of the total cell proteins in the E. coli BL21(DE3) strain for 8 transformants. The results of SDS-PAGE and Western blot analysis indicated that a cysteine protease of approximately 25 kDa (mature form) was generated. The optimal pH and temperature of the enzyme were determined to be approximately 9.5 and $35^{\circ}C$, respectively, thereby indicating that the cysteine protease is a member of the alkaline protease group. The evaluation of substrate specificity indicated that the purified protease was more active towards Arg-X or Lys-X and did not efficiently cleave the substrates with non-polar amino acids at the P1 site. The PLCP evidenced fibrinolytic activity on the plasminogen-free fibrin plate test.