• Microbiology and Biotechnology Letters
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• v.32 no.4
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• pp.297-306
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• 2004

Heat shock proteins (HSPs) were induced in cells in the thermal stress, and the HSP90 family is one of the major classes of HSPs. Gene encoding HSPs have been characterized from various mammals and piscine. We have cloned and sequenced the HSP90 cDNA from a brain cDNA library constructed from flounder (Paralichthys oliThe result of sequence analysis shows it to be the HSP90~. The nucleotide sequence of the HSP90$\beta$ was composed of 2791 long, encoding 726 amino acid residues. The flounder hsp90$\beta$ gene showed very high sequence homology with hsp90f3 of European sea bass (96.6%), zebrafish (92.9%), Atlantic salmon (92.0%) and human (89.5%). We also constructed a phylogenetic tree based on HSP90 amino acid sequences from vertebrate species. Gene-specific primers were selected and used in RT-PCR reactions to measure the basal hsp90$\beta$ mRNA. The hsp90f3 gene is constitutively expressed at a fairly high level in all examined tissues (brain, liver, kidney, muscle, and spleen). In order to express protein of flounder hsp90$\beta$ in E. coli, we used the His-tagged pETvector. Then, the expression of flounder HSP90$\beta$ was confirmed by Western blot analysis.

• Journal of Microbiology and Biotechnology
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• v.21 no.2
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• pp.176-182
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• 2011

The study objective was to prepare biodegradable branched dextran microspheres encapsulated with His-tagged interferon-alpha (BDM-hIFN-${\alpha}$) and evaluate its activity in vitro and in vivo. The glycidyl methacrylate derivatized dextrans (Dex-GMA) as precursor was primarily synthesized by substituting hydroxyl groups of either the branched or linear type of dextran with GMA. Dex-GMA microspheres loaded with hIFN-${\alpha}$ was then prepared by the water-in-water emulsion technique. In vitro release and Western blotting experiments demonstrated the retained activity of hIFN-${\alpha}$ released from branched dextran microspheres at 24 h by inducing phosphorylation of signal transducer and activator transcription-1 (STAT-1), a down-stream effector of IFN-${\alpha}$, in HepG2 cells. Animal data further revealed a peak of plasma levels of IFN-${\alpha}$ in rats injected intravenously with BDM-hIFN-${\alpha}$ at 10 min post-injection, but a sharp decline at 2 h. High plasma levels of neopterin, a plasma protein induced by IFN-${\alpha}$, were also detected in rats injected with BDM-hIFN-${\alpha}$ at 10 min post-injection. Notably, plasma levels of neopterin remained high at 4 h, but largely declined thereafter.

• Journal of Microbiology and Biotechnology
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• v.29 no.12
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• pp.1916-1924
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• 2019

Outbreaks of staphylococcal food poisoning (SFP) causing serious human diseases and economic losses have been reported globally. Furthermore, the spread of Staphylococcus aureus with increased resistance to multiple antimicrobial agents has become a major concern in the food industries and medicine. Here, we isolated an endolysin LysSAP8, as one of the peptidoglycan hydrolases, derived from the bacteriophage SAP8 infecting S. aureus. This endolysin was tagged with a 6×His at the C-terminal of the target protein and purified using affinity chromatography. LysSAP8 demonstrated lytic activity against a broad spectrum of bacteria, which included a majority of the staphylococcal strains tested in this study as well as the methicillin-resistant S. aureus (MRSA); however, no such activity was observed against other gram-positive or gram-negative bacteria. Additionally, LysSAP8 could maintain bactericidal activity until 0.1 nM working concentration and after heat treatment at 37℃ for 30 min. The ability of LysSAP8 to lyse cells under varying conditions of temperature (4-43℃), pH (3-9), and NaCl concentrations (0-1,000 mM), and divalent metal ions (Ca²⁺, Co²⁺, Cu²⁺, Mg²⁺, Mn²⁺, Hg²⁺, and Zn²⁺) was examined. At the optimized condition, LysSAP8 could disrupt approximately 3.46 log CFU/ml of the planktonic cells in their exponential phase of growth within 30 min. In this study, we have suggested that LysSAP8 could be a potent alternative as a biocontrol agent that can be used to combat MRSA.

• Journal of Microbiology and Biotechnology
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• v.22 no.4
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• pp.535-540
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• 2012

${\beta}$-D-Xylosidase (E.C. 3.2.1.37) from Bifidobacterium breve K-110, which hydrolyzes ginsenoside Ra1 to ginsenoside Rb2, was cloned and expressed in Escherichia coli. The ($His_6$)-tagged recombinant enzyme, designated as XlyBK-110, was efficiently purified using $Ni^{2+}$-affinity chromatography (109.9-fold, 84% yield). The molecular mass of XylBK-100 was found to be 55.7 kDa by SDS-PAGE. Its sequence revealed a 1,347 bp open reading frame (ORF) encoding a protein containing 448 amino acids, which showed 82% identity (DNA) to the previously reported glycosyl hydrolase family 30 of Bifidobacterium adolescentis ATCC 15703. The $K_m$ and $V_{max}$ values toward p-nitrophenyl-${\beta}$-D-xylopyranoside (pNPX) were 1.45mM and 10.75 ${\mu}mol/min/mg$, respectively. This enzyme had pH and temperature optima at 6.0 and $45^{\circ}C$, respectively. XylBK-110 acted to the greatest extent on xyloglucosyl kakkalide, followed by pNPX and ginsenoside Ra1, but did not act on p-nitrophenyl-${\alpha}$-L-arabinofuranoside, p-nitrophenyl-${\beta}$-D-glucopyranoside, or p-nitrophenyl-${\beta}$-D-fucopyranoside. In conclusion, this is the first report on the cloning and expression of ${\beta}$-D-xylosidase-hydrolyzing ginsenoside Ra1 and kakkalide from human intestinal microflora.

• Korean Journal of Microbiology
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• v.45 no.4
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• pp.332-338
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• 2009

A xylan-decomposing bacterium, HY-20, was isolated from the gut of a honeybee, Apis mellifera, and identified as Bacillus sp. The extracellular GH11 xylanase (XylP) gene (687-bp) of strain HY-20 encoded a protein of 228 amino acids with a deduced molecular mass of 25,522 Da and a calculated pI of 9.33. The primary structure of XylP was 97% identical to that of B. pumilus xylanase (GenBank accession no.: AY526092) that has not been characterized yet. The recombinant His-tagged enzyme (rXylP) overexpressed in Escherichia coli BL21 harboring pET-28a(+)/xylP was purified to electrophoretic homogeneity by cation exchange and gel permeation chromatographies. The purified enzyme exhibited the highest catalytic activity toward birchwood xylan at pH 6.5 and $50^{\circ}C$ and retained approximately 50% of its original activity when pre-incubated at $55^{\circ}C$ for 15 min. The recombinant enzyme was completely inactivated by $Hg^{2+}$ (1 mM) and N-bromosuccinimide (5 mM), while its activity was slightly stimulated by approximately 10% in the presence of $Mn^{2+}$ (1 mM), $Fe^{2+}$ (1 mM), and sodium azide (5 mM). rXylP was able to efficiently degrade various polymeric xylose-based substrates but PNP-sugar derivatives and glucose-based polymers were not susceptible to the enzyme.

• Parasites, Hosts and Diseases
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• v.50 no.1
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• pp.15-21
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• 2012

In Iran, $Plasmodium$ $vivax$ is responsible for more than 80% of the infected cases of malaria per year. Control interventions for vivax malaria in humans rely mainly on developed diagnostic methods. Recombinant $P.$ $vivax$ apical membrane antigen-1 (rPvAMA-1) has been reported to achieve designing rapid, sensitive, and specific molecular diagnosis. This study aimed to perform isolation and expression of a rPvAMA-1, derived from Iranian patients residing in an endemic area. Then, the diagnostic efficiency of the characterized Iranian PvAMA-1 was assessed using an indirect ELISA method. For this purpose, a partial region of AMA-1 gene was amplified, cloned, and expressed in pET32a plasmid. The recombinant $His-tagged$ protein was purified and used to coat the ELISA plate. Antibody detection was assessed by indirect ELISA using rPvAMA-1. The validity of the ELISA method for detection of anti-$P.$ $vivax$ antibodies in the field was compared to light microscopy on 84 confirmed $P.$ $vivax$ patients and compared to 84 non-$P.$ $vivax$ infected individuals. The ELISA cut-off value was calculated as the mean+2SD of OD values of the people living in malaria endemic areas from a south part of Iran. We found a cut-off point of OD=0.311 that showed the best correlation between the sera confirmed with $P.$ $vivax$ infection and healthy control sera. A sensitivity of 81.0% and specificity of 84.5% were found at this cut off titer. A good degree of statistical agreement was found between ELISA using rPvAMA-1 and light microscopy (0.827) by Kappa analysis.