• Environmental Analysis Health and Toxicology
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• 제29권
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• pp.18.1-18.9
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• 2014

Objectives The purpose of this study was to determine a separation method for each arsenic metabolite in urine by using a high performance liquid chromatography (HPLC)-inductively coupled plasma-mass spectrometer (ICP-MS). Methods Separation of the arsenic metabolites was conducted in urine by using a polymeric anion-exchange (Hamilton PRP X-100, $4.6mm{\times}150mm$, $5{\mu}m$) column on Agilent Technologies 1260 Infinity LC system coupled to Agilent Technologies 7700 series ICP/MS equipment using argon as the plasma gas. Results All five important arsenic metabolites in urine were separated within 16 minutes in the order of arsenobetaine, arsenite, dimethylarsinate, monomethylarsonate and arsenate with detection limits ranging from 0.15 to $0.27{\mu}g/L$ ($40{\mu}L$ injection). We used G-EQUAS No. 52, the German external quality assessment scheme and standard reference material 2669, National Institute of Standard and Technology, to validate our analyses. Conclusions The method for separation of arsenic metabolites in urine was established by using HPLC-ICP-MS. This method contributes to the evaluation of arsenic exposure, health effect assessment and other bio-monitoring studies for arsenic exposure in South Korea.

• Journal of Microbiology and Biotechnology
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• 제15권5호
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• pp.1047-1053
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• 2005

Cyclosporin A binding protein type-19 kDa peptidyl-prolyl cis/trans isomerase (PPIases, EC 5.2.1.8) of Euglena gracilis was purified and some of its biochemical characters were elucidated. Purification of the PPIase was achieved by employing a series of steps involving ammonium sulfate precipitation, Superdex G-75 gel filtration chromatography, Mono­Q anion and Mono-S cation exchange chromatographies, and Superdex S-200 gel filtration chromatography on FPLC. Purified PPIase had a specific activity of 8,250 units/mg, showing a 27-fold increase compared with that of cell-free extract of Euglena gracilis. The enzyme consisted of a single polypeptide chain with a molecular mass of 19 kDa. It showed high substrate specificity to succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, and $k_{car}/K_{m}$, for this substrate was found to be $61.19{\times}10^5/sec$. The isomer distributions were investigated at an equilibrium of seven different peptide substrates, varying Xaa in Suc-Ala-Xaa-Pro-Phe-p-nitroanilide in dimethylsulfoxide. The cis/trans equilibrium constants were estimated to be from 0.14 (Ile) to 0.63 (Gly), which correspond to $12.00\%\;to\;38.52\%$ of the cis population, respectively, under experimental condition. The enzyme was highly sensitive to the immunosuppressive ligand cyclosporin A, but not to other immunosuppressants such as FK506 and rapamycin. Thus, it appears to belong to the class of cyclophilin.

• Journal of Microbiology and Biotechnology
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• 제25권9호
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• pp.1449-1459
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• 2015

A novel proteolytic enzyme with fibrinolytic activity, FSP3, was purified from the recently isolated Streptomyces sp. P3, which is a novel bacterial strain isolated from soil. FSP3 was purified to electrophoretic homogeneity by ammonium sulfate precipitation, anion exchange, and gel filtration. FSP3 is considered to be a single peptide chain with a molecular mass of 44 kDa. The maximum activity of the enzyme was observed at 50℃ and pH 6.5, and the enzyme was stable between pH 6 and 8 and below 40℃. In a fibrin plate assay, FSP3 showed more potent fibrinolytic activity than urokinase, which is a clinical thrombolytic agent acting as a plasminogen activitor. The activity was strongly inhibited by the serine protease inhibitor PMSF, indicating that it is a serine protease. Additionally, metal ions showed different effects on the activity. It was significantly suppressed by Mg²⁺ and Ca²⁺ and completely inhibited by Cu²⁺, but slightly enhanced by Fe²⁺. According to LC-MS/MS results, its partial amino acid sequences are significantly dissimilar from those of previously reported fibrinolytic enzymes. The sequence of a DNA fragment encoding FSP3 contained an open reading frame of 1287 base pairs encoding 428 amino acids. FSP3 is a bifunctional enzyme in nature. It hydrolyzes the fibrin directly and activates plasminogen, which may reduce the occurrence of side effects. These results suggest that FSP3 is a novel serine protease with potential applications in thrombolytic therapy.

• Journal of Microbiology and Biotechnology
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• 제19권12호
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• pp.1557-1564
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• 2009

Human CDX2 is known as a caudal-related homeodomain transcription factor that is expressed in the intestinal epithelium and is important in differentiation and maintenance of the intestinal epithelial cells. The caudal-related homeobox proteins bind DNA according to a helix-turn-helix structure, thereby increasing the structural stability of DNA. A cancer-tumor suppressor role for Cdx2 has been shown by a decrease in the level of the expression of Cdx2 in colorectal cancer, but the mechanism of transcriptional regulation has not been examined at the molecular level. We developed a large-scale system for expression of the recombinant, novel CDX2, in Escherichia coli. A highly purified and soluble CDX2 protein was obtained in E. coli strain BL21(DE3)RIL and a hexahistidine fusion system using Ni-NTA affinity column, anion exchange, and gel filtration chromatographies. The identity and secondary structure of the purified CDX2 protein were confirmed by MALDI-TOF MS, Western blot, and a circular dichroism analyses. In addition, we studied the DNA-binding activity of recombinant CDX2 by ELISA experiment and isolated human CDX2-binding proteins derived from rat cells by an immobilized GST-fusion method. Three CDX2-binding proteins were found in the gastric tissue, and those proteins were identified to the homeobox protein Hox-D8, LIM homeobox protein 6, and SMC1L1 protein.

• 한국수산과학회지
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• 제8권3호
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• pp.171-176
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• 1975

개불, Urechis unicinctus, 건조중의 핵산관계물질의 변화를 음이온교환칼럼크로마토그래피로 실험하여 다음과 같은 결과를 얻었다. 살아있는 개불에는 AMP가 건물량기준으로 $6.8\mu\;mole/g$ 로서 가장 함량이 높았고, IMP는 존재하지 않았다. 건조중 ATP, ADP, AMP는 감소하고 inosine과 hypoxanthine은 증가하였으나, AMP이외의 다른 pattern은 함량이 적고 개불에서와 마찬가지로 건조한 개불에 있어서도 AMP가 건물량기준으로 $5\mu\;mole/g$로서 함량이 가장 많았다.

• Journal of Microbiology and Biotechnology
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• 제22권9호
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• pp.1237-1244
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• 2012

Agarase genes of non-marine agarolytic bacterium Cellvibrio sp. were cloned into Escherichia coli and one of the genes obtained using HindIII was sequenced. From nucleotide and putative amino acid sequences (713 aa, molecular mass; 78,771 Da) of the gene, designated as agarase AgaA, the gene was found to have closest homology to the Saccharophagus degradans (formerly, Microbulbifer degradans) 2-40 aga86 gene, belonging to glycoside hydrolase family 86 (GH86). The putative protein appears to be a non-secreted protein because of the absence of a signal sequence. The recombinant protein was purified with anion exchange and gel filtration columns after ammonium sulfate precipitation and the molecular mass (79 kDa) determined by SDS-PAGE and subsequent enzymography agreed with the estimated value, suggesting that the enzyme is monomeric. The optimal pH and temperature for enzymatic hydrolysis of agarose were 6.5 and $42.5^{\circ}C$, and the enzyme was stable under $40^{\circ}C$. LC-MS and NMR analyses revealed production of a neoagarobiose and a neoagarotetraose with a small amount of a neoagarohexaose during hydrolysis of agarose, indicating that the enzyme is a ${\beta}$-agarase.

• Journal of Microbiology and Biotechnology
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• 제22권11호
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• pp.1540-1548
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• 2012

Manganese peroxidase (MnP) was isolated from the culture filtrate of the wood log mushroom Stereum ostrea (S. ostrea), grown on Koroljova medium, and then purified by ammonium sulfate [70% (w/v)] fractionation, DEAE-cellulose anion exchange chromatography, and Sephadex G-100 column chromatography, with an attainment of 88.6-fold purification and the recovery of 22.8% of initial activity. According to SDS-PAGE the molecular mass of the MnP was 40 kDa. The optimal pH and temperature were found to be 4.5 and $35^{\circ}C$, respectively. The enzyme was stable even after exposure to a pH range of 4.5 to 6.0, and at temperatures of up to $35^{\circ}C$ at a pH of 4.5 for 1h. The $K_m$ and $V_{max}$ values for the substrate phenol red were found to be $8{\mu}m$ and 111.14 U/mg of protein, respectively. The MnP also oxidized other substrates such as guaiacol, DMP, and veratryl alcohol. Sodium azide, EDTA, SDS, $Cu^{2+}$, and $Fe^{2+}$, at 1-5 mM, strongly inhibited enzyme activity, whereas $Ca^{2+}$ and $Zn^{2+}$ increased enzyme activity. The participation of the purified enzyme in the decolorization of dyes suggests that S. ostrea manganese peroxidase could be effectively employed in textile industries.

• Bulletin of the Korean Chemical Society
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• 제34권12호
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• pp.3817-3824
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• 2013

Various separation modes in HPLC, such as anion exchange (AE), reversed-phase (RP), and affinity (AF) chromatography were examined for the separation of selenium species in human blood serum and urine. While RP- and AE-HPLC were mainly used for the separation of small molecular selenium species, double column AF-HPLC achieved the separation of selenoproteins in blood serum efficiently. Further, the effluent of AF-HPLC was enzymatically hydrolyzed and then analyzed with RP HPLC for selenoamino acid study. The versatility of the hybrid technique makes the in-depth study of selenium species possible. For quantification, post column isotope dilution (ID) with $^{78}Se$ spike was performed. ORC ICP/MS (octapole reaction cell inductively coupled plasma/mass spectrometry) was used with 4 mL $min^{-1}$ Hydrogen as reaction gas. In urine sample, inorganic selenium and SeCys were identified. In blood serum, selenoproteins GPx, SelP and SeAlb were detected and quantified. The concentration for GPx, SelP and SeAlb was $22.8{\pm}3.4\;ng\;g^{-1}$, $45.2{\pm}1.7\;ng\;g^{-1}$, and $16.1{\pm}2.2\;ng\;g^{-1}$, respectively when $^{80}Se/^{78}Se$ was used. The sum of these selenoproteins ($84.1{\pm}4.4\;ng\;g^{-1}$) agrees well with the total selenium concentration measured with the ID method of $87.0{\pm}3.0\;ng\;g^{-1}$. Enzymatic hydrolysis of each selenium proteins revealed that SeCys is the major amino acid for all three proteins and SeMet is contained in SeAlb only.

• 한국식품과학회지
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• 제31권4호
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• pp.1120-1123
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• 1999

본 연구는 멸치 자건품의 선상 가공시 유출되는 자숙액 중에 다량으로 함유되어 있는 타우린을 분리, 정제하고자 하였다. 원료 자숙액 중의 타우린 함량은 112.5 mg/100g으로서 총유리아미노산 함량의 38.8%를 차지하였다. 이 자숙액을 Amberlite IR-120과 Amberlite IR-400 column에 순차적으로 적용하여 수율이 79.2%, 순도가 89.9%인 타우린을 얻었다. 또한 이를 다시 에탄올로 정제한 결과, 수율이 29.5%, 순도가 98.1%인 고순도 타우린 결정을 얻을 수 있었다.

• KSBB Journal
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• 제29권3호
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• pp.131-138
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• 2014

In this study, we isolated two novel chitinase producing bacterial strains from yellow loess samples collected from Jullanamdo province. The chitinase producing bacteria were isolated based on the zone size of clearance in the chitin agar plates. Both of them were gram positive, rod ($2{\sim}3{\times}0.3{\sim}0.4{\mu}m$), spore-forming, and motility positive. They were facultative anaerobic, catalase positive and hydrolyzed starch, gelatin, and casein. From the 16s rRNA gene sequence analysis, the isolates were labeled as Bacillus licheniformis KYLS-CU01 and B. licheniformis KYLS-CU02. The isolates showed higher extracellular chitinase activities than B. licheniformis ATCC 14580 as a control. The optimum temperature and pH for chitinase production were $40^{\circ}C$ and pH 7.0, respectively. Response Surface Methodology (RSM) was used to optimize the culture medium for efficient production of the chitinase. Under this optimal condition, 1.5 times higher chitinase activity of B. licheniformis KYLS-CU02 was obtained. Extracellular chitinases of the two isolates were purified through ammonium sulfate precipitation and anion-exchange DEAE-cellulose column chromatography. The specific activities of purified chitinase from B. licheniformis KYLS-CU01 and B. licheniformis KYLS-CU02 were 7.65 and 5.21 U/mg protein, respectively. The molecular weights of the two purified chitinases were 59 kDa. Further, the purified chitinase of B. licheniformis KYLS-CU01 showed high antifungal activity against Fusarium sp.. In conclusion, these two bacterial isolates can be used as a biopesticide to control pathogenic fungi.