• Journal of Microbiology and Biotechnology
• /
• 제23권8호
• /
• pp.1060-1069
• /
• 2013

Genome organization near cyclomaltodextrinases (CDases) was analyzed and compared for four different hyperthermophilic archaea: Thermococcus, Pyrococcus, Staphylothermus, and Thermofilum. A gene (CL1_0884) encoding a putative CDase from Thermococcus sp. CL1 (tccd) was cloned and expressed in Escherichia coli. TcCD was confirmed to be highly thermostable, with optimal activity at $85^{\circ}C$. The melting temperature of TcCD was determined to be $93^{\circ}C$ by both differential scanning calorimetry and differential scanning fluorimetry. A size-exclusion chromatography experiment showed that TcCD exists as a monomer. TcCD preferentially hydrolyzed ${\alpha}$-cyclodextrin (${\alpha}$-CD), and at the initial stage catalyzed a ring-opening reaction by cleaving one ${\alpha}$-1,4-glycosidic linkage of the CD ring to produce the corresponding single maltooligosaccharide. Furthermore, TcCD could hydrolyze branched CDs (G1-${\alpha}$-CD, G1-${\beta}$-CD, and G2-${\beta}$-CD) to yield significant amounts (45%, 40%, and 46%) of isomaltooligosaccharides (panose and $6^2$-${\alpha}$-maltosylmaltose) in addition to glucose and maltose. This enzyme is one of the most thermostable maltogenic amylases reported, and might be of potential value in the production of isomaltooligosaccharides in the food industry.

• Journal of Pharmaceutical Investigation
• /
• 제31권3호
• /
• pp.191-196
• /
• 2001

Biodegradable polymeric microspheres were studied for their usefulness as carriers for the delivery of vaccine antigens. However, protein antigen could be denatured during microencapsulation processes due to the exposure to the organic phase and stress condition of cavitation and shear force. Therefore this study was carried out to re-evaluate the degree of protein denaturation during microencapsulation with poly(lactide-co-glycolide) (PLGA) copolymer. PLGA microspheres containing ovalbumin (OVA), prepared by W/O/W multiple emulsification method, were suspended in pH 7.4 PBS and incubated with shaking at $37.5^{\circ}C$. Drug released medium was collected periodically and analyzed for protein contents by micro-BCA protein assay. In order to evaluate the protein integrity, release medium was subjected to the analyses of SDS-PAGE and size exclusion chromatography (SEC). And enzyme-linked immunosorbent assay (ELISA) was introduced to measure the immunoreactivity of entrapped OVA and to get an insight into the three-dimensional structure of epitope. The structures of entrapped protein were not affected significantly by the results of SDS-PAGE and SEC. However, immunoreactivity of released antigen was varied, revealing the possibility of protein denaturation in some microspheres when it was evaluate by ELISA method. Therefore, in order to express the degree of protein denaturation, antigenicity ratio (AR) was obtained as follows: amount of immunoreactivity of OVA/total amount of OVA released ${\times}100(%)$. ELISA method was an efficient tool to detect a protein denaturation during microencapsulation and the comparison of AR values resulted in more accurate evaluation for immunoreactivity of entrapped protein.

• IMMUNE NETWORK
• /
• 제4권2호
• /
• pp.94-99
• /
• 2004

Background: We previously reported that ginsan, a polysaccharide extracted from Panax ginseng had an immunostimulatory activity such as mitogenic activity, activation of macrophages and killer cells, and production of a variety of cytokines which resulted in antitumor and antiseptic effects. We further purified $\alpha$-(1$\longrightarrow$6)-glucan and $\beta$-(2$\longrightarrow$6)-fructan from the ginsan with size exclusion and ion-exchange column chromatography successively. In this study, we performed the structure-based activity of ginsan by comparison with known polysacchrides such as $\beta$-glucan, curdlan, laminarin, levan, dextran, lentinan and OK-432. Methods: To investigate the immunostimulatory activity of several polysaccharide compounds, we investigated the stimulation of lymphocytes proliferation, the generation of activated killer cells and the secretion of nitrites from activated macrophages. Results: Of polysaccharides tested, curdlan and ginsan stimulated lymphocyte proliferation, suggesting that the molecular weight and composition of polysaccharide are dependent on the mitogenic activity. The production of nitric oxide was significantly increased in curdlan, levan, ginsan and its fraction, indicating that fructan has also capacity to activate macrophages and may devote to kill pathogens. In addition, the activation of macrophages was seemed to be independent of molecular weight of polysaccharide. The generation of AK cells was exhibited in order of curdlan, OK-432> F1, ginsan, F3> levan> etc. The AK activity may be dependent on molecular weight and composition of polysaccharides. Conclusion: Unfortunately, purified polysaccharide from ginsan were less active on immunostimulatory activity than mixed compounds of polysaccharides. From the viewpoint of structure and activity relationships, we found several characteristic features.

• 동아시아식생활학회지
• /
• 제4권3호
• /
• pp.59-65
• /
• 1994

이 실험에서는 소의 유즙에 있는 성장인자를 세포배양을 통해 확인하고 크로마토그래피를 사용하여 분리 정제하였다. 5%의 우유를 포함하는 배지를 FBS(fetal bovine serum)를 포함한 배지와 비교했을때 Africal green monkey kidney cell의 성장 촉진 효과가 비슷하게 나타났으며, 우유의 성장인자를 분리 정제한 결과 FBS배지에서보다 2,000배 이상의 성장 촉진 효과가 나타났다. 이 성장인자는 hydrophobic column(phenyl-sepharose)과 gel-filtration column(sephadex G-100)을 사용하여 분리했으며 그 결과 milk-deriven growth factor는 phenyl-sepharose에서 50% ethylene glycol step에서 용출되므로 hydrophobic protein임이 증명되었고 size exclusion column chromatography로 부터 분자량이 100,000-15,0000 범위의 고분자 물질임이 밝혀졌다. 또한 우유의 성장인자는 매우 다양하며 그중 본 실험으로부터 정제된 물질이 주요 성장인자로 밝혀졌다.

• Membrane and Water Treatment
• /
• 제11권4호
• /
• pp.257-274
• /
• 2020

There are many previous review articles are available to summarize either the characterization methods of effluent organic matter (EfOM) or the individual control treatment options. However, there has been no attempt made to compare in parallel the physicochemical treatment options that target the removal of EfOM from biological treatments. This review deals with the recent progress on the characterization of EfOM and the novel technologies developed for EfOM treatment. Based on the publications after 2010, the advantages and the limitations of several popularly used analytical tools are discussed for EfOM characterization, which include UV-visible and fluorescence spectroscopy, Fourier transform infrared spectroscopy (FTIR), size exclusion chromatography (SEC), and Fourier transform-ion cyclotron resonance-mass spectrometry (FT-ICR-MS). It is a recent trend to combine an SEC system with various types of detectors, because it can successfully track the chemical/functional composition of EfOM, which varies across a continuum of different molecular sizes. FT-ICR-MS is the most powerful tool to detect EfOM at molecular levels. However, it is noted that this method has rarely been utilized to understand the changes of EfOM in pre-treatment or post-treatment systems. Although membrane filtration is still the preferred method to treat EfOM before its discharge due to its high separation selectivity, the minimum requirements for additional chemicals, the ease of scaling up, and the continuous operation, recent advances in ion exchange and advanced oxidation processes are greatly noteworthy. Recent progress in the non-membrane technologies, which are based on novel materials, are expected to enhance the removal efficiency of EfOM and even make it feasible to selectively remove undesirable fractions/compounds from bulk EfOM.

• 한국물환경학회지
• /
• 제25권3호
• /
• pp.419-424
• /
• 2009

Characteristics of humic substances on the changes in dissolved organic matter (DOM) characteristics by biodegradation was investigated using three types of the artificial water samples composed of glucose and Suwannee River fulvic acid (SRFA). Some selected DOM characteristics including the specific UV absorbance (SUVA), the synchronous fluorescence spectra and the molecular weight (MW) were compared for the artificial water samples before and after 28-day microbial incubation. The changes of the DOM characteristics were minimal for SRFA during the incubation whereas they were significant for glucose. SUVA, dissolved organic carbon (DOC)-normalized fluorescence intensity, and MW values of glucose increased, suggesting that such labile organic compounds could be exclusively transformed into more humidified materials by biodegradation. For glucose-SRFA mixture, the selected DOM characteristics were greater than those estimated using the assumption that the individual changes of either glucose or SRFA are conservative for the mixture of the two materials. Our results suggest that the presence of humic substances (HS) may lead to the enhancement of the formation of refractory organic materials during biodegradation of labile compounds. Detailed analyses of size exclusion chromatography (SEC) revealed that the enhancement occurred for the DOM mixture with a MW range between 500 Da to 4000 Da.

• 한국수산과학회지
• /
• 제38권5호
• /
• pp.286-290
• /
• 2005

Oxytocin (OT)-related peptide, isotocin was purified from the brain extract of conger eel (Conger myriaster) using reverse-phase, ion-exchange and size exclusion high performance liquid chromatography (HPLC). The sequence of the peptide, with a molecular weight of 967.30 Da, was determined as Cys-Tyr-Ile-Ser-Asn­Cys-Pro-Ile-Gly-$NH_2$, where the Cys between 1st and 6th residues made an intramolecular disulfide bridge by the automated amino acid sequence analysis and MALDI-TOF mass spectrometry. The sequence was confirmed by identical elution with the purified and synthetic peptide using the HPLC system. As a result of homology investigation, the primary structure of this peptide was the same as that of OT -superfamily member, isotocin. The synthetic peptide showed a contractile activity at a minimal effective concentration of $10^{-7}M$ on the intestinal smooth muscle of goldfish (Carassius auratus).

• 한국자기공명학회논문지
• /
• 제19권3호
• /
• pp.106-111
• /
• 2015

Helicobacter pylori (H. pylori) survives in acidic and fluctuating pH conditions of the stomach. The pH effect on H. pylori proteins is important for the advanced understanding of its evolution and viability, although this bacterium has the molecular machinery that neutralizes the acidic condition. HP1492 is known as a conserved NifU-like protein from H. pylori. NifU is a nitrogen fixation protein that mediates the transfer of iron-sulfur (Fe-S) cluster to iron-sulfur proteins like ferredoxin. Commonly, the monomeric reduced state of NifU can be converted to the dimeric oxidized state by intermolecular disulfide bond formation. Because it remains unclear that HP1492 actually behaves as known NifU protein, we first found that this protein can adopt both oxidized and reduced forms using size exclusion chromatography. Circular dichroism experiment showed that HP1492 is relatively well-structured at pH 6.5, compared to other pH conditions. On the basis of the backbone resonance assignment of HP1492, we further characterized the residues that are sensitive to pH using NMR spectroscopy. These residues showing large chemical shift changes could be mapped onto the secondary structure of the protein. Our results could provide the foundation for structural and biophysical studies on a wide spectrum of NifU proteins.

• KSBB Journal
• /
• 제24권5호
• /
• pp.458-462
• /
• 2009

황산 수용액에 재생 피브로인을 녹이고 가열하여 황산을 반응물과 동시에 촉매로 사용하여 황산-피브로인을 제조 하였다. 황산의 농도 및 반응온도를 달리하여 얻어진 시료로부터 적외선 흡광 스펙트럼, UV 흡광 스펙트럼, NMR 및 GPC를 사용하여 반응조건에 따른 황산기의 도입 정도 및 피브로인 분자의 분해 정도를 분석하였다. 황산-피브로인의 수율은 5%의 황산을 사용하여 $60^{\circ}C$에서 반응시킬 때 가장 높았으며 적외선 흡광 스펙트럼의 $1109\;cm^{-1}$의 피크가 나타내는 도입된 황산기의 량은 황산의 농도가 증가하면 같이 증가하였다. 단백질에 도입된 황산기는 피브로인 분자를 주로 구성하고 있는 세린 및 타이로신의 hydroxyl 그룹과 반응하여 O-sulfate ester를 형성할 것으로 예상되는데, 이는 274 nm에서의 UV 흡광도의 감소 및 $3300\;cm^{-1}$의 적외선 흡광 스펙트럼의 감소가 관찰되는 결과와 일치하였다. GPC 분석을 통하여 황산-피브로인이 가수 분해되어 원래 피브로인의 분자량 보다 분자량이 작으며 동시에 가수분해된 저분자 황산 피브로인 펩타이드와 덜 가수분해된 비교적 분자량이 큰 펩타이드로 구성되어 있음이 확인되고 있다.

• Journal of Microbiology and Biotechnology
• /
• 제17권9호
• /
• pp.1513-1520
• /
• 2007

[ ${\beta}$ ]-1,3-Glucans enhance immune reactions such as antitumor, antibacterial, antiviral, anticoagulatory, and wound healing activities. ${\beta}$-1,3-Glucans have various functions depending on the molecular weight, degree of branching, conformation, water solubility, and intermolecular association. The molecular weight of the soluble glucan was about 15,000 as determined by a high-performance size exclusion chromatography. From the infrared (IR) and $^{13}C$ NMR analytical data, the purified soluble glucan was found to exclusively consist of ${\beta}$-D-glucopyranose with 1,3 linkage. We tested the immunestimulating activities of the soluble ${\beta}$-1,3-glucan extracted from Agrobacterium sp. R259 KCTC 1019 and confirmed the following activities. IFN-$_{\gamma}$ and each cytokines were induced in the spleens and thymus of mice treated with soluble ${\beta}$-1,3-glucan. Adjuvant effect was observed on antibody production. Nitric oxide was synthesized in monocytic cell lines treated with ${\beta}$-1,3-glucan. The cytotoxic and antitumor effects were observed on various cancer cell lines and ICR mice. These results strongly suggested that this soluble ${\beta}$-1,3-glucan could be a good candidate for an immune-modulating agent.