• Molecules and Cells
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• 제25권1호
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• pp.112-118
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• 2008

Laccases are multicopper-containing oxidases that catalyze the oxidation of many aromatic compounds with concomitant reduction of oxygen to water. Interest in this enzyme has arisen in many fields of industry, including detoxification, wine stabilization, paper processing, and enzymatic conversion of chemical intermediates. In this study, we cloned a laccase gene (GLlac1) from the white-rot fungus Ganoderma lucidum. The cloned gene consists of 4,357 bp, with its coding region interrupted by nine introns, and the upstream region has putative CAAT and TATA boxes as well as several metal responsive elements (MREs). We also cloned a full-length cDNA of GLlac1, which contains an uninterrupted open reading frame (ORF) of 1,560 bp coding for 520 amino acids with a putative 21-residue signal sequence. The DNA and deduced amino acid sequences of GLlac1 were similar but not identical to those of other fungal laccases. GLlac1 was released from the cells when expressed in P. pastoris, and had high laccase activity. In addition, GLlac1 conferred antioxidative protection from protein degradation, and thus may be useful in bio-medical applications.

• Journal of Microbiology and Biotechnology
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• 제20권4호
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• pp.712-717
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• 2010

Cytochrome P450 enzymes (P450s) are involved in the synthesis of a wide variety of valuable products and in the degradation of numerous toxic compounds. The P450 BM3 (CYP102A1) from Bacillus megaterium was the first P450 discovered to be fused to its redox partner, a mammalian-like diflavin reductase. Here, we report the development of a whole-cell biocatalyst using ice-nucleation protein (Inp) from Pseudomonas syringae to display a hemeand diflavin-containing oxidoreductase, P450 BM3 (a single, 119-kDa polypeptide with domains of both an oxygenase and a reductase) on the surface of Escherichia coli. The surface localization and functionality of the fusion protein containing P450 BM3 were verified by flow cytometry and measurement of enzymatic activities. The results of this study comprise the first report of microbial cell-surface display of a heme- and diflavin-containing enzyme. This system should allow us to select and develop oxidoreductases containing heme and/or flavins into practically useful whole-cell biocatalysts for extensive biotechnological applications, including selective synthesis of new chemicals and pharmaceuticals, bioconversion, bioremediation, live vaccine development, and biochip development.

• Preventive Nutrition and Food Science
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• 제15권1호
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• pp.57-66
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• 2010

Passion fruit fiber pectin gels represent a new alternative pectin source with potential for food and non-food applications on a commercial scale. Pectic polysaccharides were extracted from passion fruit (Passiflora edulis) fiber using citric acid as a clean catalyst and autoclaved for 20 to 60 min at $121^{\circ}C$. The best condition of pectin yield with the highest molecular weight was obtained with 1.0% of citric acid (250 mg/g dry passion fruit fiber pectin) for 20 min of autoclaving. Spectroscopic analyses by Fourier transform infrared, enzymatic degradation reactions, and ion-exchange chromatography assays showed that passion fruit pectin extracted for 20 min was homogeneous high methoxylated pectin (70%). Gel permeation analysis confirmed that the pectin extract obtained by autoclaving by 20 min showed higher molecular weights than those autoclaved for 40 and 60 min. Passion fruit pectin extracted for 20 min was enzymatically modified with fungal pectinmethylesterase to create restructured gels. Short autoclave treatment (20 min) with citric acid as extractant resulted in a significant increase of gel strength, improving pectin extraction in terms of functionality. The treatment of solubilized material (pectic polysaccharides) in the presence of insoluble material (cellulose and hemicellulose) with pectinmethylesterase and calcium led to the creation of a stiffer passion fruit fiber pectin gel, while syneresis was not observed.

• BMB Reports
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• 제34권3호
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• pp.219-224
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• 2001

To identify the antioxidative peptides in the gelatin hydrolysate of bovine skin, the gelatin was hydrolyzed with serial digestions in the order of Alcalase, pronase E, and collagenase using a three-step recycling membrane reactor. The second enzymatic hydrolysate (hydrolyzed with pronase E) was composed of peptides ranging from 1.5 to 4.5 kDa, and showed the highest antioxidative activity, as determined by the thiobarbituric acid method. Three different peptides were purified from the second hydrolysate using consecutive chromatographic methods. This included gel filtration on a Sephadex G-25 column, ion-exchange chromatography on a SP-Sephadex C-25 column, and high-performance liquid chromatography on an octadecylsilane chloride column. The isolated peptides were composed of 9 or 10 amino acid residues. They are: Gly-Glu-Hyp-Gly-Pro-Hyp-Gly-Ala-Hyp (PI), Gly-ProHyp-Gly-Pro-Hyp-Gly-Pro-Hyp-Gly (PII), and Gly-ProHyp-Gly-Pro-Hyp-Gly-Pro-Hyp (PIII), as characterized by Edman degradation and fast-atom bombardment mass spectrometry. The antioxidative activities of the purified peptides were measured using the thiobarbituric acid method, and the cell viability with a methylthiazol tetrazolium assay The results showed that PII had potent antioxidative activity on peroxidation of linoleic acid. Moreover, the cell viability of cultured liver cells was significantly enhanced by the addition of the peptide. These results suggest that the purified peptide, PII, from the gelatin hydrolysate of bovine skin is a natural antioxidant, which has potent antioxidative activity.

• BMB Reports
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• 제48권2호
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• pp.91-96
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• 2015

Cells express several antioxidant enzymes to scavenge reactive oxygen species (ROS) responsible for oxidative damages and various human diseases. Therefore, antioxidant enzymes are considered biomedicine candidates. Among them, extracellular superoxide dismutase (SOD3) had showed prominent efficacy against asthma and inflammation. Despite its advantages as a biomedicine, the difficulty in obtaining large quantity of active recombinant human SOD3 (rhSOD3) has limited its clinical applications. We found that a significant fraction of over-expressed rhSOD3 was composed of the inactive apo-enzyme and its potency against inflammation depended on the rate of metal incorporation. Also, purified rhSOD3 was unstable and lost its activity very quickly. Here, we suggest an ideal preparative method to express, purify, and store highly active rhSOD3. The enzymatic activity of rhSOD3 was maximized by incorporating metal ions into rhSOD3 after purification. Also, albumin or polyethylene glycol prevented rapid inactivation or degradation of rhSOD3 during preparative procedures and long-term storage.

• Food Science and Biotechnology
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• 제16권5호
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• pp.728-733
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• 2007

Bamboo grass, Sasa quelpaertensis, is a native plant to Jeju Island, Korea. The leaves of Sasa plants are widely used in traditional Korean medicine to treat inflammation-related diseases. We investigated the effect of hot water extract from Sasa quelpaertensis leaves (HWE-SQ) on nitric oxide (NO) production and nuclear $factor-{\kappa}B\;(NF-{\kappa}B)$ activation in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. HWE-SQ inhibited LPS-induced NO production and inducible NO synthase (iNOS) protein expression in a dose-dependent manner. Reporter gene assays indicated that HWE-SQ decreases LPS-induced $NF-{\kappa}B$ transcriptional activation. However, HWE-SQ did not affect the phosphorylation and degradation of inhibitory ${\kappa}B{\alpha}\;(1{\kappa}B{\alpha})$. HWE-SQ also directly inhibited iNOS enzyme activity in a dose-dependent manner. These results suggest that HWE-SQ suppresses NO synthesis in macrophages by attenuating $NF-{\kappa}B-mediated$ iNOS protein expression and inhibiting iNOS enzymatic activity, thereby implicating a mechanism by which HWE-SQ is able to ameliorate inflammation-related diseases by limiting excessive or prolonged NO production in pathological events.

• The Korean Journal of Physiology and Pharmacology
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• 제13권3호
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• pp.195-200
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• 2009

Zinc released from excited glutamatergic neurons accelerates amyloid ${\beta}$ (A ${\beta}$) aggregation, underscoring the therapeutic potential of zinc chelation for the treatment of Alzheimer's disease (AD). Zinc can also alter A ${\beta}$ concentration by affecting its degradation. In order to elucidate the possible role of zinc influx in secretase-processed A ${\beta}$ production, SH-SY5Y cells stably expressing amyloid precursor protein (APP) were treated with pyrrolidine dithiocarbamate (PDTC), a zinc ionophore, and the resultant changes in APP processing were examined. PDTC decreased A ${\beta}$ 40 and A ${\beta}$ 42 concentrations in culture media bathing APP-expressing SH-SY5Y cells. Measuring the levels of a series of C-terminal APP fragments generated by enzymatic cutting at different APP-cleavage sites showed that both ${\beta}$-and ${\alpha}$-cleavage of APP were inhibited by zinc influx. PDTC also interfered with the maturation of APP. PDTC, however, paradoxically increased the intracellular levels of A ${\beta}$ 40. These results indicate that inhibition of secretase-mediated APP cleavage accounts -at least in part- for zinc inhibition of A ${\beta}$ secretion.

• Nutrition Research and Practice
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• 제1권4호
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• pp.285-290
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• 2007

The leukocyte recruitment and transmigration across the endothelial barrier into the vessel wall are crucial steps in atherosclerosis. Leukocyte trafficking on the endothelium is elicited by induction of endothelial adhesion molecules, and its transmigration is mediated by degradation of basement membrane proteins through enzymatic activity of matrix metalloproteinases (MMP). The current study investigated whether resveratrol, a polyphenol present in grapes and red wine, was capable of inhibiting leukocyte adhesion to tumor necrosis factor (TNF)-${\alpha}$-activated endothelium. It was found that resveratrol inhibited the TNF-${\alpha}$-activated endothelial expression of vascular cell adhesion molecule-1 in a dose-dependent manner. In addition, resveratrol hampered THP-1 monocyte adhesion to activated endothelial cells. This study further examined whether resveratrol interfered with transendothelial migration of leukocytes. The MMP-2 gelatinolytic activity of endothelial cells was enhanced by TNF-${\alpha}$, which was attenuated by an addition of ${\geq}25{\mu}M$ resveratrol. In addition, 25 ${\mu}M$ resveratrol mitigated the MMP-9 activity of THP-1 cells, followed by a marked inhibition of transendothelial migration. These results demonstrated that resveratrol suppressed monocyte adhesion and migration induced by TNF-${\alpha}$ through modulating expression of adhesion molecules and gelatinolytic activity of MMP. These findings suggest that dietary resveratrol may be therapeutic agent for inhibiting leukocyte recruitment into the subendothelium during inflammatory atherosclerosis.

• Journal of Microbiology
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• 제35권2호
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• pp.109-116
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• 1997

An extracellular proteinase of Candida albicans was purified by a combination of 0~75% ammonium sulfate precipitation, DEAE Sepharose Fast Flow ion exchange chromatography, and Sephacryl S-200 HR molecular sieve chromatography. Its mlecular weight was approximately 41 kDa on SDS-PAGE and isoelectric point was 4.4. The enzyme was inhibited by pepstain A. Optimum enzyme activity ranged from pH 2.0 to 3.5 with its maximum at pH 2.5 and a temperature of 45$^{\circ}C$. The addition of divalent cations, $Ca^{2+}$, Zn$^{2+}$ and $Mg^{2+}$, resulted in no significant inhibition of enzymatic activity. However, some inhibitory effects were observed by Fe$^{2+}$, Ag$^{2+}$ and Cu$^{2+}$. With BSA as substrate, an apparent $K_m$ was determined to be 7$\times$10$^{-7}$ M and $K_i$, using pepstatin A as an inhibitor, was 8.05$\times$10$^{-8}$ M. N-terminal amino acid sequence was QAVPVTLXNEQ. Degradation of BSA and fibronectin was shown but not collagen, hemoglobin, immunoglobulin G, or lysozyme. The enzyme preferred peptides with Glu and Leu at the P$_1$ position, but the enzyme activity was highly reduced when the P$_2$ position was phe or pro. This enzyme showed antigenicity against sera of patients with candidiasis.

• Parasites, Hosts and Diseases
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• 제32권4호
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• pp.231-242
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• 1994

자연 감염된 가재에서 폐흡충의 피낭유충을 분리하고 개에 경구 감염시켜 성충을 얻었다. 폐흡충 성충의 조효소를 ion-exchange chromatography, affinity chromatography와 gel filtration chromatoglaphy를 실시하여 cysteine proteinase를 순수 정제하였다. 이들 효소의 생화학적 특성과 분해능을 관찰하였으며. 효소면역전기영동이적법을 이용하여 순수 정제한 효소의 항원성을 관찰하였다. 정제된 효소는 저분자 합성기질인 CBZ-arg-arg-AFC 보다 CBZ-phe-arg-AFC에서 높은 활성을 보였으며. 이들 효소는 thiol-dependent이었다. 정제된 효소 및 조효소의 최적 pH는 5.5이었고. 최적 mole 농도는 0.1 M(0.1 M sodium citrate, pH 5.5)이었고, 이들 효소는 $4^{\circ}C$에서 48시간 동안 80%의 안정성을 보였다 정제된 효소의 native 분자량은 20.000 dalton이었고, SDS- PAGE상에 나타난 분자량은 17,500 dalton이었다 정제된 효소는 cysteine proteinase 특이 억제 인자인 E-64, lodoacetic acid, NEM에 의해 활성이 완전히 억제되었으며, serine proteinase, aspartic proteinase 및 metallo proteinase 특이 억제인자에 의해 활성이 억제되지 않았다. 정제된 효소는 collagen(Type I)과 hemoglobin을 분해하였고, 효소면역전기영동이적법으로 정제된 효소의 항원성을 확인하였다.