• Parasites, Hosts and Diseases
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• v.31 no.3
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• pp.259-268
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• 1993

When activity of peroxidase in auld Pnrqfonimn westermqni was monitored using o-dianisidine and $H_2O_2$ as substrates, its specific activity was 1.5 times higher In excretory-secretory product (ESP) than in crude extract. The one was purified by two purification steps of Sephacryl S-300 Superfine gel permeation and DEAE-Trisacryl M anion exchange chromatographies. Its activity increased 16.9 fold with 32.3% recovery. The enzyme was inhibited totally by 1 millimoles of dithiothreitol (DTT), 2-mercaptoethanol and azide. Molecular mass was 16 kDa in reducing SDS-polyacrylamide gel electrophoresis (PAGE) or 19 kDa in TSK-Blue gel filtration high performance liquid chromatography (HPLC). respectively. Special staining for peroxidase by diaminobenzidine on SDS-PAGE confirmed the activity. The peroxidase was less reactive to a paragonimiasis serum when observed by SDS-PAGE/immunoblot. In addition, specific activities of superoxide dismutase (SOD) and catalase were also identified in the ESP. High activities of these antioxidant enzymes in ESP indicate that they are parts of defense mechanisms against reactive oxygen intermediates from host.

• Journal of Life Science
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• v.16 no.7 s.80
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• pp.1133-1140
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• 2006

Human HtrA1 (High temperature requirement protein A1) is a homologue of the E. coli periplasmic serine protease HtrA. A recent study has demonstrated that HtrA1 is a serine protease involved in processing of insulin like growth factor binding protein (ICFBP), indicating that it serves as an important regulator of IGF activity. Additionally, several lines of evidence suggest a striking correlation between proteolytic activity of HtrA1 serine protease and the pathogenesis of several diseases; however, physiological roles of HtrA1 remain to be elucidated. We used the pGEX bacterial expression system to develop a simple and rapid method for purifying HtrA1, and the recombinant HtrA1 protein was utilized to investigate the optimal conditions in executing its proteolytic activity. The proteolytically active HtrA1 was purified to approximately 85% purity, although the yield of the recombinant HtrA1 protein was slightly low $460{\mu}g$ for 1 liter E. coli culture). Using in vitro endoproteolytic cleavage assay, we identified that the HtrA1 serine protease activity was dependent on the enzyme concentration and the incubation time and that the best reaction temperature was $42^{\circ}C$ instead of $37^{\circ}C$. We arbitrary defined one unit of proteolytic activity of the HtrA1 serine protease as 200nM of HtrA1 that cleaves half of $5{\mu}M\;of\;{\beta}-casein$ during 3 hr incubation at $37^{\circ}C$. Our study provides a method for generating useful reagents to investigate the molecular mechanisms by which HtrA1 serine protease activity contributes in regulating its physiological function and to identify natural substrates of HtrA1.

• KSBB Journal
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• v.19 no.1
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• pp.50-56
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• 2004

The aim of this work was to investigate the purification and characterization dixoygenases isolated from Delftia sp. JK-2, which could utilize aniline, benzoate, and p-hydroxybenoate as sole carbon and energy source. Catechol 1,2-dioxygenase (C1, 2O), catechol 2,3-dioxygenase(C2, 3O), and protocatechuate 4,5-dioxygenase(4,5-PCD) were isolated by benzoate, aniline, and p-hydroxybenzoate. In initial experiments, several characteristics of C1 ,2O, C2, 3O, and 4,5-PCD separated with ammonium sulfate precipitation, DEAE-sepharose, and Q-sepharose were investigated. Specific activity of C1 ,2O, C2, 3O, and 4,5-PCD were approximately 3.3 unit/mg, 4.7 unit/mg, and 2.0 unit/mg. C1 ,2O and C2, 3O demonstrated their enzyme activities to other substrates, catechol and 4-methylcatechol. 4,5-PCD showed the specific activity to the only substrate, protocatechuate, but the substrates(e.g., catechol, 3-methylcatechol, 4-methylcatechol, 4-chlorocatechol, 4-nitrocatechol) did not show any specific activities in this work. The optimum temperature of C1, 2O, C2, 3O, and 4,5-PCD were 30$^{\circ}C$, and the optimal pHs were approximately 8, 8, and 7, respectively. Ag$\^$+/, Hg$\^$+/, Cu$\^$2+/ showed inhibitory effect on the activity of C1, 2O and C2, 3O, but Ag$\^$+/, Hg$\^$+/, Cu$\^$2+/, Fe$\^$3+/ showed inhibitory effect on the activity of 4,5-PCD. Molecular weight of the C1, 2O, C2, 3O, and 4,5-PCD were determined to approximately 60 kDa,35 kDa, and 62 kDa by SDS-PAGE.

• Microbiology and Biotechnology Letters
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• v.40 no.1
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• pp.57-65
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• 2012

Antifungal compounds from Bacillus polyfermenticus CJ6 were purified using SPE, preparative HPLC, and reverse phase-HPLC. Antifungal compounds from B. polyfermenticus CJ6 were separated into three fractions (8, B, C) using preparative HPLC. LC/MS analysis of antifungal peaks suggested that B. polyfermenticus CJ6 produces lipopeptides; two kinds of iturin A ($C_{14}$, $C_{15}$), three kinds of surfactins ($C_{13}$, $C_{14}$, $C_{15}$), four kinds of fengycin A ($C_{14}$, $C_{15}$, $C_{16}$, $C_{17}$) and two kinds fengycin B ($C_{16}$, $C_{17}$). The antifungal activity of fraction 8, which was presumed as inturin A, was found to be stable after the pH, heat or proteolytic enzyme treatment, but it was unstable at 50-$70^{\circ}C$ for 24 hr. The antifungal activity of fraction B, which presumed as surfactins and fengycin A, was found to be stable after the heat treatment, but it was unstable in the pH 3.0 and after the protease (type I) or ${\alpha}$-chymotrypsin treatment. The antifungal activity of fraction C, which was presumed as fengycin A and B, was found to be stable in the pH 3.0-9.0 range and the heat treatment, but it was unstable with the treatment of protease (type I). The amino acid composition of the purified peaks 8-1 and 8-2 were Asx, Tyr, Gln, Pro, and Ser in a molar ratio of 3:1:1:1:1, which showed the same amino acid composition as iturin. From these results, we confirmed that antifungal compounds from B. polyfermenticus CJ6 most likely belonged to iturin A as well as surfactins and fengycins. As lipopeptides are known to act in a synergistic manner, the antifungal compounds from B. polyfermenticus CJ6 might have potential uses in biotechnology and biopharmaceutical applications.