• BMB Reports
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• 제29권2호
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• pp.175-179
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• 1996

Viral surface proteins are known to play an essential role in attachment of the virus particle to the host cell membrane. In case of the hepatitis B virus (HBV) several reports have described potential receptors on the target cell side, but no definite receptor protein has been isolated yet. As for the viral side, it has been suggested that the preS region of the envelope protein, especially the preS1 region, is involved in binding of HBV to the host cell. In this study, preS1 region was recombinantly expressed in the form of a maltose binding protein (MBP) fusion protein and used to identify and visualize the expression of putative HBV receptor(s) on the host cell. Using laser scanned confocal microscopy and by FACS analysis, MBP-preS1 proteins were shown to bind to the human hepatoma cell line HepG2 in a receptor-ligand specific manner. The binding kinetic of MBP-preS1 to its cellular receptor was shown to be temperature and time dependent. In cells permeabilized with Triton X-100 and treated with the fusion protein, a specific staining of the nuclear membrane could be observed. To determine the precise location of the receptor binding site within the preS1 region, several short overlapping peptides from this region were synthesized and used in a competition assay. In this way the receptor binding epitope in preS1 was revealed to be amino acid residues 27 to 51, which is in agreement with previous reports. These results confirm the significance of the preS1 region in virus attachment in general, and suggest an internalization pathway mediated by direct attachment of the viral particle to the target cell membrane.

• BMB Reports
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• 제28권3호
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• pp.254-260
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• 1995

Cytochrome oxidase was purified from bovine-heart mitochondria and its enzymatic properties were examined. The purified cytochrome oxidase was identified by its absorption spectrum and chromatogram through gel filtration. The specific activity, purification degree and yield of purified cytochrome oxidase were 18 nmol/mg/ml/min, 24.83 fold and 0.93%, respectively. The activity of the enzyme assayed by a ferrocytochrome $c-O_2$ system was optimized at $25^{\circ}C$ and pH 6.5. Examining the effect of nonionic detergents established that cytochrome oxidase was deactivated by Triton X-100. The oxidase was activated by Tween 80 and deactivated by Tween 20. The Michaelis constant and maximum velocity of the oxidase for ferrocytochrome c were 0.032~0.044 mM and 0.019~0.021 mM/min, respectively. After adaption to basal diet for a week, experimental diets containing 6 mg Cu/kg, or zero mg Cu/kg, or 12 mg Cu/kg were fed to a control group, a copper-free group and a copper-rich group of Sprague-Dawley rats, respectively, for 4 weeks. The specific activities assayed for the ferrocytochrome $c-O_2$ system of isolated cytochrome oxidase from the rat liver of control, copper-free, and copper-rich group were 1.00, 1.19, and 0.878 nmol/mg/ml/min, respectively. Their degrees of purification were 11.38, 10.82 and 8.78 fold, respectively. The specific activities for liver and heart mitochondrial cytochrome oxidase of copper-free/copper-rich groups assayed using the ferrocytochrome $c-O_2$ system were 81.4% and 96.4%/64.1% and 61.1%, respectively, compared with those of the control.

• Natural Product Sciences
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• 제26권3호
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• pp.207-213
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• 2020

Characterization and in vitro inhibition studies of protease inhibitor from the mushroom Pleurotus floridanus (PfTI) towards the pest Papilio demoleus is studied. The addition of 1 mM Mn²⁺, Na²⁺, Ba²⁺ and Ni ²⁺ enhanced the PfTI activity. The ICP-atomic emission spectrum showed the presence of Ca²⁺, Mg²⁺ and Zn²⁺ in the PfTI. Surfactants SDS and CTAB at a concentration of 1% reduced the PfTI activity whereas, the nonionic detergents Triton X and Tween 80 increased the activity. The inhibitory activity gradually decreased with increase in concentration of DMSO and H₂O₂. The activity was increased by dithiothreitol up to a concentration of 80 μM and inactivated at 140 μM. The activity of PMSF modified PfTI was drastically reduced to 0.234 U/mL at 4 mM concentration and similar results were obtained for modification of cysteine by N-Ethylmaleimide at slightly higher concentrations. The complex of trypsin and PfTI showed complete loss in fluorescence intensity at 343 nm compared with control. In vitro inhibition studies of PfTI with midgut proteases isolated from citrus pest P. demoleus with protease activity of 1.236 U was decreased to 0.613 U by 50 μL (0.1 mg/mL) of the inhibitor. Inhibitor was stable up to 0.04 M concentration of HCl.

• Journal of Microbiology and Biotechnology
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• 제30권8호
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• pp.1207-1213
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• 2020

Aflatoxin B1 (AFB1) is a mycotoxin produced by Aspergillus flavus (A. flavus). AFB1 is reported to have high thermal stability and is not decomposed by heat treatment during food processing. Therefore, in this study, knowing that AFB1 is metabolized by cytochrome P450 (CYP), our aim was to develop a method to detoxify A. flavus-contaminated maize, under normal temperature and pressure, using Escherichia coli expressing human CYP3A4. First, the metabolic activity of AFB1 by recombinant human CYP3A4 was evaluated. As a result, we confirmed that recombinant human CYP3A4 metabolizes 98% of AFB1. Next, we found that aflatoxin Q1, a metabolite of AFB1 was no longer mutagenic. Furthermore, we revealed that about 50% of the AFB1 metabolic activity can be maintained for 3 months when E. coli expressing human CYP3A4 is freeze-dried in the presence of trehalose. Finally, we found that 80% of AFB1 in A. flavus-contaminated maize was metabolized by E. coli expressing human CYP3A4 in the presence of surfactant triton X-405 at a final concentration of 10% (v/v). From these results, we conclude that AFB1 in A. flavus-contaminated maize can be detoxified under normal temperature and pressure by using E. coli expressing human CYP3A4.

• Journal of Microbiology and Biotechnology
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• 제19권9호
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• pp.932-945
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• 2009

Several species of Gram-positive bacteria have cell wall peptidoglycan (syn. murein) in which not all of the sugar moieties are N-acetylated. This has recently been shown to be a secondary effect, caused by the action of a peptidoglycan N-acetylglucosamine deacetylase. We have found that the opportunistic pathogen Listeria monocytogenes is unusual in having three enzymes with such activity, two of which remain in the cytoplasm. Here, we examine the enzyme (PgdA) that crosses the cytoplasmic membrane and is localized in the cell wall. We purified a hexa-His-tagged form of PgdA to study its activity and constructed a mutant devoid of functional Lmo0415 (PgdA) protein. L. monocytogenes PgdA protein exhibited peptidoglycan N-acetylglucosamine deacetylase activity with natural substrates (peptidoglycan) from both L. monocytogenes and Escherichia coli as well as the peptidoglycan sugar chain component N-acetylglucosamine, but not with N-acetylmuramic acid. As was reported recently [6], inactivation of the structural gene was not lethal for L. monocytogenes nor did it affect growth rate or morphology of the cells. However, the pgdA mutant was more prone to autolysis induced by such agents as Triton X-100 and EDTA, and is more susceptible to the cationic antimicrobial peptides (CAMP) lysozyme and mutanolysin, using either peptidoglycan muramidases or autolysis-inducing agents. The pgdA mutant was also slightly more susceptible than the wild-type strain to the action of certain beta-lactam antibiotics. Our results indicate that protein PgdA plays a protective physiological role for listerial cells.

• Journal of Microbiology and Biotechnology
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• 제19권2호
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• pp.128-135
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• 2009

Acinetohacter baumannii BD5 was isolated from waters of Baek-du mountain, and the lipase gene was cloned using a PCR technique. The deduced amino acid sequence of the lipase and lipase chaperone were found to encode proteins of 325 aa and 344 aa with a molecular mass of 35 kDa and 37 kDa, respectively. The lipase gene was cloned and expressed in Escherichia coli BL21(trxB) as an inclusion body, which was subsequently solubilized by urea, and then purified using Ni-affinity chromatography. After being purified, the lipase was refolded by incubation at $4^{\circ}C$ in the presence of a 1:10 molar ratio of lipase:chaperone. The maximal activity of the refolded lipase was observed at a temperature of $35^{\circ}C$ and pH 8.3 when p-NP caprate(C10) was used as a substrate; however, 28% of the activity observed at $35^{\circ}C$ was still remaining at $0^{\circ}C$. The stability of the purified enzyme at low temperatures indicates that it is a cold-adapted enzyme. The refolded lipase was activated by $Ca^{2+},\;Mg^{2+},\;and\;Mn^{2+}$, whereas $Zn^{2+}\;and\;Cu^{2+}$ inhibited it. Additionally, 0.1% Tween 20 increased the lipase activity by 33%, but SDS and Triton X-100 inhibited the lipase activity by 40% and 70%, respectively.

• 한국미생물·생명공학회지
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• 제36권2호
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• pp.128-134
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• 2008

Erro-prone PCR에 의해서 열안정성이 향상된 Streptomyces coelicolor A(3) 유래의 L-threonine aldolase를 Corynebacterium glutamicum R에서 과잉발현시키기 위하여 Corynebacterium용 vector plasmid인 pCRB1의 SD배열과 개시코든사이의 1염기를 제거한 고발현용 vector plasmid인 pCG-H44(2)를 구축하였다. pCG-H (2)에 의해서 형질전환된 C. glutamicum R 균주(CGH44-2)에서 L-TA를 발현시킨 결과, 기존의 Corynebacterium용 vector plasmid인 pCRB1(CGH44-1)보다 L-TA의 발현량이 높았다. L-threo-DOPS의 합성을 위한 최적조건은 $30^{\circ}C$, 0.1 M cirtric acid buffer(pH 7.0)이었으며, 0.1% TritonX-100를 첨가하였을 경우 보다 높은 활성을 보였다. 최적조건하에서 CGH44-2를 whole cell biocatalyst로 이용한 반복회분식반응에서 재조합대장균을 숙주로 이용한 경우보다 재조합Corynebacterium을 이용하였을 경우, 목적하는 L-threo-DOPS의 합성이 안정적으로 이루어졌다.

• Journal of Microbiology and Biotechnology
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• 제19권7호
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• pp.727-733
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• 2009

Heat shock protein 70 kDa (hsp70), a molecular chaperone involved in folding of nascent proteins, has been studied for its ability to activate innate and specific immunity. High purity hsp70 preparation is generally required for immunization experiments, because endotoxins and other immunologically active contaminants may affect immune responses independently of hsp70. We have developed a novel modification of E. coli-expression medium that enabled a simple two-step production and purification method for endotoxin-free recombinant hsp70. During Ni-NTA-based affinity purification of hsp70, a contaminating protein from host E. coli cells, L-glutamine-n-fructose-6-phosphate aminotransferase (GFAT), was identified. By testing various compounds, supplementation of growth medium with a GFAT metabolite,N-acetylglucosamine, was found to reduce GFAT expression and increase the total hsp70 yield five times. The new protocol is based on column purification of His-tagged hsp70 protein produced by E. coli with the modified medium, followed by endotoxin removal by Triton X-114 extraction. This approach yielded hsp70 with high purity and minimal endotoxin contamination, making the final product acceptable for immunization experiments. In summary, a simple modification of growth medium allowed production of recombinant mouse hsp70 in high yield and purity, thus compatible with immunological studies. This protocol may be useful for production of other Histagged proteins expressed in E. coli.

• Environmental Engineering Research
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• 제14권4호
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• pp.250-254
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• 2009

Contamination of microcystins, a family of heptapeptide hepatotoxins, in eutrophic water bodies is a worldwide problem. Due to their poisoning effects on animals and humans, there is a requirement to characterize and quantify all microcystins present in a sample. As microcystins are, for most part, intracellular toxins produced by some genera of cyanobacteria, lysing cyanobacterial cells to release all microcystins is considered an important step. To date, although many cell lysis methods have been used, little work has been conducted comparing the results of those different methods. In this study, various methods for cell lysis and toxin extraction from the cell lysates were investigated, including sonication, bead beating, freeze/thaw, lyophilization and lysing with TritonX-100 surfactant. It was found that lyophilization, followed by extraction with 75% methanol, was the most effective for extracting toxins from Microcystis aeruginosa cells. Another important step prior to the analysis is removing impurities and concentrating the target analyte. For these purposes, a C18 Sep-Pak solid phase extraction cartridge was used, with the percentage of the eluent methanol also evaluated. As a result, methanol percentages higher than 75% appeared to be the best eluting solvent in terms of microcystin-leucine-arginine (MC-LR) recovery efficiency for the further chromatographic and mass spectrometric analyses.

• 약학회지
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• 제48권6호
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• pp.384-390
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• 2004

Polyphenol oxidase-catalyzed oxidation of substrates (t-butylcatechol, 4-methylcatechol, chlorogenic acid, caffeic acid and pyrocatechol) were performed in the Ph range 4~8. Co ncentrations of substrate's major oxidation products were monitored by high performance liquid chromatograph. The nature and amounts of products formed were highly pH dependent. They also were ifluenced by kinds of substrates. Major oxidation product of 4-methylcatechol appeared the maxium value at pH 5, them of chlorogenic acid, caffeic acid and pyrocatechol at pH 6.0 and that of t-butylcatechol at pH 5~7. Time-dependent PPO activity was determined at $4^{\circ}C\;and\;30^{\circ}C$. PPO extracted by phosphate buffer containing triton X-114 (t-PPO) was more stable than PPO by phosphate buffer (b-PPO). The result of electrophoresis, at first PPO was showed only a band at 48 kd. After 1~3 days a partial degrade band was appeared in b-PPO and three partial degrade bands in t-PPO. No activity band was appeared in PPOs at $30^{\circ}C$ and b-PPO at $4^{\circ}C$ after 4 days. And a band (37 kDa) in t-PPO was remained finally and disappered. PPO from Perillae leaves has two activity bands at 48 and 37 kDa in previous paper. It was supposed that PPO in the leaves of Perilla frutescens was a protein having one molecular weight as 48 kDa. And 37 kDa protein, relatively proteolysis-resistant, was a proteolyzed form of a major form.