• Journal of Microbiology and Biotechnology
• /
• 제21권8호
• /
• pp.808-817
• /
• 2011

Because of its elevated cellulolytic activity, the filamentous fungus Trichoderma harzianum has a considerable potential in biomass hydrolysis applications. Trichoderma harzianum cellobiohydrolase I (ThCBHI), an exoglucanase, is an important enzyme in the process of cellulose degradation. Here, we report an easy single-step ion-exchange chromatographic method for purification of ThCBHI and its initial biophysical and biochemical characterization. The ThCBHI produced by induction with microcrystalline cellulose under submerged fermentation was purified on DEAE-Sephadex A-50 media and its identity was confirmed by mass spectrometry. The ThCBHI biochemical characterization showed that the protein has a molecular mass of 66 kDa and pI of 5.23. As confirmed by smallangle X-ray scattering (SAXS), both full-length ThCBHI and its catalytic core domain (CCD) obtained by digestion with papain are monomeric in solution. Secondary structure analysis of ThCBHI by circular dichroism revealed ${\alpha}$- helices and ${\beta}$-strands contents in the 28% and 38% range, respectively. The intrinsic fluorescence emission maximum of 337 nm was accounted for as different degrees of exposure of ThCBHI tryptophan residues to water. Moreover, ThCBHI displayed maximum activity at pH 5.0 and temperature of $50^{\circ}C$ with specific activities against Avicel and p-nitrophenyl-${\beta}$-D-cellobioside of 1.25 U/mg and 1.53 U/mg, respectively.

• 대한약침학회지
• /
• 제19권1호
• /
• pp.37-44
• /
• 2016

Objectives: This study examined the relative efficacies of a derivative of betulinic acid (dBA) and its poly (lactide-co-glycolide) (PLGA) nano-encapsulated form in A549 lung cancer cells in vivo and in co-mutagen [sodium arsenite (SA) + benzo[a]pyrene (BaP)]-induced lung cancer in mice in vivo. Methods: dBA was loaded with PLGA nanoparticles by using the standard solvent displacement method. The sizes and morphologies of nano-dBA (NdBA) were determined by using transmission electron microscopy (TEM), and their intracellular localization was verified by using confocal microscopy. The binding and interaction of NdBA with calf thymus deoxyribonucleic acid (CT-DNA) as a target were analyzed by using conventional circular dichroism (CD) and melting temperature (Tm) profile data. Apoptotic signalling cascades in vitro and in vivo were studied by using an enzyme-linked immunosorbent assay (ELISA); the ability of NdBA to cross the blood-brain barrier (BBB) was also examined. The stage of cell cycle arrest was confirmed by using a fluorescence-activated cell-sorting (FACS) data analysis. Results: The average size of the nanoparticles was ~ 110 nm. Confocal microscopy images confirmed the presence of NdBA in the cellular cytoplasm. The bio-physical properties of dBA and NdBA ascertained from the CD and the Tm profiles revealed that NdBA had greater interaction with the target DNA than dBA did. Both dBA and NdBA arrested cell proliferation at G0/G1, NdBA showing the greater effect. NdBA also induced a greater degree of cytotoxicity in A549 cells, but it had an insignificant cytotoxic effect in normal L6 cells. The results of flow cytometric, cytogenetial and histopathological studies in mice revealed that NdBA caused less nuclear condensation and DNA damage than dBA did. TEM images showed the presence of NdBA in brain samples of NdBA fed mice, indicating its ability to cross the BBB. Conclusion: Thus, compared to dBA, NdBA appears to have greater chemoprotective potential against lung cancer.

• Journal of Microbiology and Biotechnology
• /
• 제27권5호
• /
• pp.1023-1031
• /
• 2017

The conformational change of cellular prion protein ($PrP^C$) to its misfolded counterpart, termed $PrP^{Sc}$, is mediated by a hypothesized cellular cofactor. This cofactor is believed to interact directly with certain amino acid residues of $PrP^C$. When these are mutated into cationic amino acid residues, $PrP^{Sc}$ formation and prion replication halt in a dominant negative (DN) manner, presumably due to strong binding of the cofactor to mutated $PrP^C$, designated as DN PrP mutants. Previous studies demonstrated that plasminogen and its kringle domains bind to PrP and accelerate $PrP^{Sc}$ generation. In this study, in vitro binding analysis of kringle domains of plasminogen to Q167R DN mutant PrP (PrPQ167R) was performed in parallel with the wild type (WT) and Q218K DN mutant PrP (PrPQ218K). The binding affinity of PrPQ167R was higher than that of WT PrP, but lower than that of PrPQ218K. Scatchard analysis further indicated that, like PrPQ218K and WT PrP, PrPQ167R interaction with plasminogen occurred at multiple sites, suggesting cooperativity in this interaction. Competitive binding analysis using $\small{L}$-lysine or $\small{L}$-arginine confirmed the increase of the specificity and binding affinity of the interaction as PrP acquired DN mutations. Circular dichroism spectroscopy demonstrated that the recombinant PrPs used in this study retained the ${\alpha}$-helix-rich structure. The ${\alpha}$-helix unfolding study revealed similar conformational stability for WT and DN-mutated PrPs. This study provides an additional piece of biochemical evidence concerning the interaction of plasminogen with DN mutant PrPs.

• 한국잠사곤충학회지
• /
• 제48권1호
• /
• pp.11-15
• /
• 2006

천잠 견단백질을 이용한 비의류용 소재개발을 위한 기초 연구의 일환으로 천잠 가수분해 견단백질 분말을 제조하여 그 구조 및 별 특성을 살펴보고 중금속 함량을 분석하였다. 1. 천잠 고치를 가수분해하여 제조한 분말의 평균분자량은 430 정도였으며 아미노산 조성 분석 결과 일반적인 천잠 견피브로인의 조성과 유사하였다. 2. 천잠 분말의 X-선 회절 분석 결과 $2{\theta}=20.34^{\circ}$ 부근과 $31.5^{\circ}$ 부근에서 강한 회절 피크를 보였으며 원이색분석 결과 220nm의 피크와 215nm의 shoulder를 나타내어 ${\alpha}-helix$와 ${\beta}-sheet$ 구조임을 알 수 있었다. 3. 시차열분석 및 열중량분석 결과 천잠 가수분해 분말은 $250^{\circ}C$ 부근에서 열분해 흡열피크를 보였으며 $270^{\circ}C$ 부근에서 열분해에 의한 최대 중량감소가 나타났다. 4. 천잠 분말에서는 수은, 비소 등 인체 독성이 강한 중금속을 관찰할 수 없었으므로 기능성 소재 개발을 위한 재료로 천잠 가수분해 분말을 활용할 수 있을 것으로 사료된다.

• 한국어병학회지
• /
• 제9권1호
• /
• pp.65-77
• /
• 1996

Tachyplesin I은 투구게로부터 단리된 항균성 펩티드이다. 인지질막에 대한 tachyplesin I의 작용 메카니즘을 조사하기 위해서 tachyplesin I 및 5개의 유도체를 액상법으로 합성하였다. 합성한 5개의 유도체는 [$Phe^2$]-tachyplesin I, [$Phe^{8,13}$]-techyplesin I, S-S결합을 가지지 않는 [$Cys(Acm)^{3,7,12,16}$]-tachyplesin I 및 [$Cys(Acm)^{3,7,12,16}$]-tachyplesin I 의 단편인 7(Acm)과 10(Acm)이다. 원편광이색성 (CD) 스펙트럼에서 tachyplesin I은 완충액에서 역평행 $\beta$-구조를 취하며 산성 지질막하에서는 완충액보다 약간 불규칙적인 구조를 가진다. Carboxyfluorescein 누출 실험결과 tachyplesin은 중성 및 산성 지질막과 강하게 상호 작용 하였다. 또한 형광 실험하에서는 펩티드의 소수성 부분이 인지질막의 내부에 존재하였다. 7(Acm) 및 10(Acm) 유도체를 제외한 모든 펩티드들은 lipopolysaccharide결합에 있어 거의 유사한 활성을 나타냈었다. 따라서 지질이중막은 tachyplesin I을 안정한 $\beta$-구조로부터 덜 규칙적인 구조로 구조적 변화를 유도한다고 여겨진다.

• KSBB Journal
• /
• 제14권6호
• /
• pp.651-654
• /
• 1999

재조합 인성장호르몬을 사용하여 in vitro 재접힘 공정(풀림, 희석에 의한 공기 중 산화, 그리고 투석)을 수행하였다. 표면소수성이 풀림-재접힘 공정에 중요한 역할을 한다는 것을 형광값의 변화를 통하여 알 수 있었다. 변성제의 intermediate 농도는 Urea와 Gu-HCl 경우 하나의 peak로 SDS와 Sarkosyl의 경우 두개의 peak로 나타났다. 형광값의 변화 중 특이한 점은 Urea의 경우 공기 중 산화와 투석 중의 후반부에 형광값이 증가한다는 것이다. 따라서 공기 중 산화도중 형광값이 증가하기 전에 투석을 시킨 결과 형광값이 증가를 막을 수 있었다. 아직 이 원인에 대해 자세히 알 수 없지만 계속 실험 중에 있다. 이번 실험에서 표면소수성 변화와 연관시켜 fluorescence를 이황화결합에 의한 산화된 형태를 알아보기 위한 방법으로 RP-HPLC를 마지막으로 단백질의 2차원적인 구조를 알아 보기 위해 CD를 사용하였다. CD측정 결과 Gu-HCl보다 SDS의 경우 ${\alpha}$-helices의 파괴가 더 많음을 볼 수 있었다. 재접힘된 rhGH는 본래의 2차원적 구조의 90%이상을 얻을 수 있었다. 이 실험이 기지는 의의는 이 모든 실험결과를 토대로 단백질 재접힘을 모니터링 하였다는 점이다. 즉, 형광값의 변화를 통하여 형광값이 증가하는 것은 표면 소수성이 증가함을 보이는 것으로 단백질의 풀림이 일어난 것이고 3차원적 구조가 깨지고 2차원 구조를 알아 볼 수 있는 ${\alpha}$-helices의 감소를 의미하였다. 이와는 반대로 형광값이 감소하는 것을 통해 재접힘이 일어남을 알 수 있었고, 이러한 결과를 바탕으로 단백질의 재접힘 공정의 변화과정을 형광값을 통하여 모니터링 할 수 있었다. 또한 이 실험의 목적 단백질은 rhGH이지만 다른 단백질에 적용이 될 경우 단백질 재접힘 과정을 수시로 모니터링하고 상태를 예측할 수 있으므로 산업현장에서 소량의 sample로 재접힘 상태를 쉽고 빠르게 판단할 수 있을 것이다. 단백질 재접힘 과정에서 이러한 개념의 성공적 도입은 단백질 회수 수율을 높임으로써 생물분리공정 분야의 기술 발전에 이바지 하리라 사료된다.

• BMB Reports
• /
• 제40권3호
• /
• pp.315-324
• /
• 2007

The novel $\alpha$-amylase purified from locally isolated strain, Bacillus sp. KR-8104, (KRA) (Enzyme Microb Technol; 2005; 36: 666-671) is active in a wide range of pH. The enzyme maximum activity is at pH 4.0 and it retains 90% of activity at pH 3.5. The irreversible thermoinactivation patterns of KRA and the enzyme activity are not changed in the presence and absence of $Ca^{2+}$ and EDTA. Therefore, KRA acts as a $Ca^{2+}$-independent enzyme. Based on circular dichroism (CD) data from thermal unfolding of the enzyme recorded at 222 nm, addition of $Ca^{2+}$ and EDTA similar to its irreversible thermoinactivation, does not influence the thermal denaturation of the enzyme and its Tm. The amino acid sequence of KRA was obtained from the nucleotide sequencing of PCR products of encoding gene. The deduced amino acid sequence of the enzyme revealed a very high sequence homology to Bacillus amyloliquefaciens (BAA) (85% identity, 90% similarity) and Bacillus licheniformis $\alpha$-amylases (BLA) (81% identity, 88% similarity). To elucidate and understand these characteristics of the $\alpha$-amylase, a model of 3D structure of KRA was constructed using the crystal structure of the mutant of BLA as the platform and refined with a molecular dynamics (MD) simulation program. Interestingly enough, there is only one amino acid substitution for KRA in comparison with BLA and BAA in the region involved in the calcium-binding sites. On the other hand, there are many amino acid differences between BLA and KRA at the interface of A and B domains and around the metal triad and active site area. These alterations could have a role in stabilizing the native structure of the loop in the active site cleft and maintenance and stabilization of the putative metal triad-binding site. The amino acid differences at the active site cleft and around the catalytic residues might affect their pKa values and consequently shift its pH profile. In addition, the intrinsic fluorescence intensity of the enzyme at 350 nm does not show considerable change at pH 3.5-7.0.

• 한국미생물생명공학회:학술대회논문집
• /
• 한국미생물생명공학회 2001년도 Proceedings of 2001 International Symposium
• /
• pp.149-153
• /
• 2001

To investigate the mode of bactericidal action for antimicrobial peptide, pediocin, synthetic and mutant pediocins were prepared by direct chemical synthesis. Native pediocin was purified from Pedio-coccus acidilactici M and its conformational structure and bactericidal functions were analyzed and compared to synthetic pediocin. Schematic mode of pediocin actions, how pediocin binds on the target cell membrane, penetrates and makes tunnel are proposed. For these purposes, primary and secondary structures of pediocin was analyzed and disulfide bond assignment was also done. The pediocin purified from P. acidilactici M had high effective bactericidal ability against gram positive bacteria, especially Listeria monocytogenes and was very stable at extreme pHs and even at high temperatures such as autoclaving temperature (121$^{\circ}C$). Pediocin was consisted of 44 amino acids with four cysteines. Novel synthetic peptides were achieved by solid phase peptide synthesis(SPPS) method. To explain the function of cysteine in C-terminal region, mutant pediocin, Ped[C24A＋C44A], was synthesized and their structural and biological functions were analyzed. Second mutant pediocin, Ped[KllE], was prepared to explain the function of lysine at 11 of N-terminal part of pediocin, especially loop of $\beta$-sheet, and to predict the initial binding site of pediocin. The native and synthetic pediocins was showed random coil conformation by spectropolarimetry in moderate conditions. This conformation was observed in extreme conditions such as high temperature and low and high pHs, also. Circular dichroism(CD) data also showed the existence of $\beta$-turn structure in N-terminal part both native and synthetic pediocins. A structural model for pediocin predicts that 18 amino acids in the N-terminal part of the peptide assume a three-strand $\beta$-sheet conformation. This random coil in C-terminal part of pediocin was converted to folding structure, helix structure, in nonpolar solvents such as alcohol and TFE. The disulfide bond between $^{9}$ Cys and $^{14}$ Cys was concrete and inevitable, however, evidences of disulfide bond between $^{24}$ Cys and $^{44}$ Cys was not. Data of Ped[C24A＋C44A], pediocin mutant showed that $^{44}$ Cys was required during killing the target cells but not inevitable, since Ped[C24A＋C44A] still have bactericidal activity but much less than native pediocin. Another pediocin mutant, Ped[KllE], had still bactericidal activity, was controversial to propose that positive charge like as $^{11}$ Lys in loop or hinge in bacteriocin bound or helped to binding to microorganism with electrostatic interaction between cell membrane especially teichoic acid and positive amino acid nonspecifically. The conformation of pediocin among native, synthetic and mutant pediocins did not show big difference. The conformations between oxidized and reduced pediocin were almost similar regardless of native or synthetic.

• Food Science and Biotechnology
• /
• 제18권1호
• /
• pp.228-233
• /
• 2009

This study was conducted to examine the changes in the molecular structure and physiological activities of silk fibroin by gamma irradiation. The results of gel permeation chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the molecular weight of fibroin was increased depending upon the irradiation dose. Secondary structure of fibroin determined by using circular dichroism revealed that the ratio of $\alpha$-helix was increased up to 10 kGy and then decreased depending upon the irradiation dose. Whereas, the ratio of $\beta$-sheet, $\beta$-turn, and random coil were decreased and then increased with an alteration in the $\alpha$-helix secondary conformation. The 2.2-diphenyl-1-picryl-hydrazil (DPPH) radical scavenging activity of fibroin was increased by gamma irradiation at 5 kGy, but was decreased above 10 kGy depending upon the irradiation dose. Also, the inhibition activities of tyrosinase and melanin synthesis of fibroin were increased by gamma irradiation. These results indicated that gamma irradiation could be used as an efficient method to make fibroin more suitable for the development of functional foods and cosmetics.

• 한국축산식품학회지
• /
• 제37권1호
• /
• pp.123-133
• /
• 2017

The development of a new manufacturing process, a two-step temperature treatment, to modulate the physicochemical properties of nanoparticles including the size is critical. This is because its physicochemical properties can be key factors affecting the cellular uptake and the bioavailability of bioactive compounds encapsulated in nanoparticles. The aims of this study were to produce (beta-lactoglobulin) ${\beta}-lg$ nanoparticles and to understand how two-step temperature treatment could affect the formation and physicochemical properties of ${\beta}-lg$ nanoparticles. The morphological and physicochemical properties of ${\beta}-lg$ nanoparticles were determined using atomic force microscopy and a particle size analyzer, respectively. Circular dichroism spectroscopy was used to investigate the secondary structure of ${\beta}-lg$. The surface hydrophobicity and free thiol groups of ${\beta}-lg$ were increased with a decrease in sub-ambient temperature and an increase in mild heat temperature. As sub-ambient temperature was decreased, a decrease in ${\alpha}-helical$ content and an increase in ${\beta}-sheet$ content were observed. The two-step temperature treatment firstly involved a sub-ambient temperature treatment from 5 to $20^{\circ}C$ for 30 min, followed secondly by a mild heat temperature treatment from 55 to $75^{\circ}C$ for 10 min. This resulted in the production of spherically-shaped particles with a size ranging from 61 to 214 nm. Two-way ANOVA exhibited the finding that both sub-ambient and mild heat temperature significantly (p<0.0001) affected the size of nanoparticles. Zeta-potential values of ${\beta}-lg$ nanoparticles were reduced with increasing mild heat temperature. In conclusion, two-step temperature treatment was shown to play an important role in the manufacturing process - both due to its inducement of the conformational changes of ${\beta}-lg$ during nanoparticle formation, and due to its modulation of the physicochemical properties of ${\beta}-lg$ nanoparticles.