• Microbiology and Biotechnology Letters
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• v.36 no.3
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• pp.182-188
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• 2008

A hyperthermophilic bacteria (strain HJ6) was isolated from a hot springs located in the Arima-cho, Hyogo, Japan. The cells were long-rod type ($2-4{\mu}m$), about $0.4{\mu}m$ in diameter. The pH and temperature for optimal growth were 6.5 and $80^{\circ}C$, respectively. Phylogenetic analysis based on the 16S rDNA sequence and biochemical studies indicated that HJ6 belonged to the genus Thermus thermophilus (Tt). The gene encoding the Trehalose synthase (TS) was cloned and sequenced. The open reading frame (ORF) of the TtTS gene was composed of 2,898 nucleotides and encoded a protein (975 amino acids) with a predicted molecular weight of 110.56 kDa. The deduced amino acid sequence of TtTS showed 99% and 83% identities to the Thermus caldophilus TS and Meiothermus ruber TS, respectively. TtTS gene was expressed in Escherichia coli cells, and the recombinant protein was purified to homogeneity. The optimal temperature and pH for Trehalose synthase activity were found to be $80^{\circ}C$ and 7.5, respectively. The half-life of heat inactivation was about 40 min at $90^{\circ}C$. The maximum trehalose conversion rate of maltose into trehalose by the enzyme increased as the substrate concentration increased, and reached 55.7% at the maltose concentration of 500 mM, implying that the enzyme conversion was dependent of the substrate concentration.

• The Korean Journal of Physiology and Pharmacology
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• v.5 no.4
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• pp.287-297
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• 2001

Contraction of smooth muscle is initiated by an increase in cytosolic $Ca^{2+}$ leading to activation of $Ca^{2+}$/ calmodulin-dependnet myosin light chain (MLC) kinase and phosphorylation of MLC. The types of contraction and signaling mechanisms mediating contraction differ depending on the region. The involvement of these different mechanisms varies depending on the source of $Ca^{2+}$ and the kinetic of $Ca^{2+}$ mobilization. $Ca^{2+}$ mobilizing agonists stimulate different phospholipases $(PLC-{\beta},\;PLD\;and\;PLA_2)$ to generate one or more $Ca^{2+}$ mobilizing messengers $(IP_3\;and\;AA),$ and diacylglycerol (DAG), an activator of protein kinase C (PKC). The relative contributions of $PLC-{\beta},\;PLA_2$ and PLD to generate second messengers vary greatly between cells and types of contraction. In smooth muscle cell derived form the circular muscle layer of the intestine, preferential hydrolysis of $PIP_2$ and generation of $IP_3$ and $IP_3-dependent\;Ca^{2+}$ release initiate the contraction. In smooth muscle cells derived from longitudinal muscle layer of the intestine, preferential hydrolysis of PC by PLA2, generation of AA and AA-mediated $Ca^{2+}$ influx, cADP ribose formation and $Ca^{2+}-induced\;Ca^{2+}$ release initiate the contraction. Sustained contraction, however, in both cell types is mediated by $Ca^{2+}-independent$ mechanism involving activation of $PKC-{\varepsilon}$ by DAG derived form PLD. A functional linkage between $G_{13},$ RhoA, ROCK, $PKC-{\varepsilon},$ CPI-17 and MLC phosphorylation in sustained contraction has been implicated. Contraction of normal esophageal circular muscle (ESO) in response to acetylcholine (ACh) is linked to $M_2$ muscarinic receptors activating at least three intracellular phospholipases, i.e. phosphatidylcholine-specific phospholipase C (PC-PLC), phospholipase D (PLD) and the high molecular weight (85 kDa) cytosolic phospholipase $A_2\;(cPLA_2)$ to induce phosphatidylcholine (PC) metabolism, production of diacylglycerol (DAG) and arachidonic acid (AA), resulting in activation of a protein kinase C (PKC)-dependent pathway. In contrast, lower esophageal sphincter (LES) contraction induced by maximally effective doses of ACh is mediated by muscarinic $M_3$ receptors, linked to pertussis toxin-insensitive GTP-binding proteins of the $G_{q/11}$ type. They activate phospholipase C, which hydrolyzes phosphatidylinositol bisphosphate $(PIP_2),$ producing inositol 1, 4, 5-trisphosphate $(IP_3)$ and DAG. $IP_3$ causes release of intracellular $Ca^{2+}$ and formation of a $Ca^{2+}$-calmodulin complex, resulting in activation of myosin light chain kinase and contraction through a calmodulin-dependent pathway.

• Microbiology and Biotechnology Letters
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• v.26 no.6
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• pp.523-528
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• 1998

We isolated microorganism secreting antimicrobial substance from tomato and identified as Enterococcus faecium. This substance was completely inactivated by pretense treatment and retained activity after catalase treatment. This result indicated that the antimicrobial activity of this substance was due to proteinaceous substance known as bacteriocin. The bacteriocin inhibited growth of Gram positive bacteria, such as Listeria monocytogenes, Leuconostoc mesenteroides, Lactobacillus plantarum, Streptococcus agalactiae, Streptococcus pyrogenes, and Gram negative bacteria, such as Pseudomonas aeruginosa. Purification of the bacteriocin was achieved by ethanol precipitation, ion exchange chromatography on CM Sepharose CL-6B, and gel filtration on Sephacryl S-100 HR. After these purification steps, the specific activity of the bacteriocin was increased 35.8 fold compared with culture broth. Purified bacteriocin was shown single band on SDS-PAGE and molecular weight was estimated 51 kDa. The residual activity of this bacteriocin was 3.3% at 10$0^{\circ}C$ for 60 min, and this bacteriocin was stable at pH 2~7.

• Microbiology and Biotechnology Letters
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• v.17 no.4
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• pp.358-364
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• 1989

A crystalline alkaline pretense- producing Streptomyce sp. YSA-130 was isolated from soil in alkaline medium(pH 10.5). The optimum culture condition of Streptomyce sp. YSA-130 for the production of alkaline protease was as follows; 2.0% soluble starch, 1.0% soytone, 0.3% $K_2$HPO$_4$, 0.02% MgSO$_4$.7$H_2O$, 0.8% Na$_2$CO$_3$, pH 10.5, 3$0^{\circ}C$, and 12 hr. The alkaline pretense from the culture broth of Streptomyce sp. YSA-130 was purified about 24 folds by ammonium sulfate precipitation , dialysis, DEAE-cellulose ion exchange chromatography, gel filtration on Sephadex G-15 and crystallization. Optimum temperature and pH of purified enzyme were 6$0^{\circ}C$, and 11.5. Temperature and pH stability of purified enzyme were 5$0^{\circ}C$, and 5.5-12.0. Calcium ion was effective to stabilize the enzyme at higher temperature. The molecular weight of the purified enzyme was approximately 30,000. The purified enzyme was inactivated by diisopropyl flurophosphate(DFP) but not affected by metal ion, EDTA, sulfhydryl reagent and stable detergent.

• Journal of Life Science
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• v.16 no.2 s.75
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• pp.175-179
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• 2006

A bacterium that produced a large amount of poly-$\gamma$-glutamate (PGA) was isolated from the compost and designated as Bacillus subtilis CH-10. The optimum temperature and pH for PGA production were at $37^{\circ}C$ and 7.5, respectively. The maximum amount of PGA production (18.84 mg/ml) was obtained when it was grown in a medium containing 3% L-glutamate and 5% sucrose at $37^{\circ}C$ with shaking. The result that the L-glutamate significantly induced PGA production indicates that it produces a PGA by the glutamate dependent manner. Some properties of the PGA obtained at different times of cultivation were investigated by SDS-PAGE and ninhydrin analysis. The PGA production was elongated along with cultivation time and maximum amount was achieved at 96 h. Average molecular weight of PGA was estimated to be 1100 kDa by FDNB method.

• Journal of Life Science
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• v.16 no.2 s.75
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• pp.245-252
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• 2006

A bacterial strain, identified as Staphylococcus aureus JJ-11, producing collagenase was isolated out of 40 persons having skin troubles. S. aureus JJ-11 produced collagenase optimally in the media containing 1.5%(w/v) gelatin, 1%(w/v) yeast extract, 0.4%(w/v) $K_2HPO_4$, 0.005%(w/v) $NiSO_4{\cdot}6H_2O$ at $37^{\circ}C$ for 18 hrs. The collagenase produced by Staphylococcus aureus JJ-11 was purified at 6.66-folds purity through application of chromatography with Amberlite IRA-900 and Sephacryl S-300 HR columns. The molecular weight of the partially purified enzyme was estimated to be 62 kDa by SDS-PAGE. The protein exhibited optimum enzymatic activity at pH 7.0, and showed a stable activity at pH 4-8. The optimum temperature for collagenase was at $37^{\circ}C$, and activity was maintained upto $40^{\circ}C$. The enzyme activity was slightly elevated in the presence of divalents such as, $Fe^{2+},\;Co^{2+}\;and\;Ba^{2+}$ However, the activity was inhibited in the presence of $Sr^{2+}\;or\;Hg^{2+}$. The inhibition of activity by O-phenanthroline and EDTA suggested that the enzyme may contain metal which is required for activity. The enzyme showed the highest activity when insoluble collagen (type I) was, used as a substrate.

• Journal of Life Science
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• v.13 no.1
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• pp.90-98
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• 2003

An allium rhizobacterium Serratia plymuthica AL-1 was previously selected as a biocontrol agent of allium white rot. The chitinase from S. plymuthica AL-1 produced in medium containing colloidal chitin was purified by ammonium sulfate precipitation (40~70%), affinity adsorption, column chromatography on DEAE-sephadex A-50 and sephadex C-200 gel filtration. The enzyme was purified 10.8-fold with a yield of 7.3% from the starting culture broth. The purified chtinase gave a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis, it's molecular weight was estimated to be 55 kDa. The optimum pH and temperature of the purified enzyme were pH 5.5 and $55^{\circ}C$, respectively and it is stable up to $50^{\circ}C$ and maintains around 90% of its activity for 60min. The enzyme were activated by $Ca^{2+}$, $Mn^{2+}$ and $Mg^{2+}$ and inhibited by $Cu^{2+}$, SDS, $\rho$-CMB, MIA, respectively. The purified chitinase showed broad spectrum of antifungal activities against plant pathogenic fungi Sclerotium cepivoruin, Alternana alternnta, Colletotrichum glceosporioidrs, Phoma sp., Sclerotinia sclerotiorum, Stemphylium solani, Fusarium oxysporium f. sp. niveum but rarely inhibited Phytophthora capsici and Pythium ultimum.. The purified chitinase from S. plymuthica AL-1 caused swelling, lysis, deceleration and degradation of the hyphal tips of S. sczerotiorum causing allium white rot. It suggest that S. prymuthica AL-1 chitinase play an important part in the bifunctional chitinase / lysozyme activity.

• Korean Journal of Veterinary Service
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• v.20 no.1
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• pp.103-125
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• 1997

An epidemiologic study on pleuropneumonia in the slaughter pigs(Chonan and Asan area, Chungnam province, Korea) during the period of January 1994 through December 1995 was conducted. Isolation of A pleuropneumoniae was attempted in 425 pigs with pneumonic lesions. Biochemical properties, antimicrobial susceptibility, serotypes and pathogenicity of isolated A pleuropneumoniae were investigated. In addition, outer membrane protein(OMP) of the Isolates were extracted to determine its properties and immunogenicity in both mice and piglets The results obtained through this study were summarized as followed ; 1. Of 3, 395 slaughter pigs, pleuropneumonia was observed in 425 pigs(10.6%). A pleuropneumoniae was isolated from 22 pigs(5.2%) out of 425 pigs with pneumonic lesions. The biochemical properties of all isolates were same as those of reference A pleuropneumoniae strain. Among 22 isolates, 9, 1 and 12 isolates were serovar 2, 3 and 5, respectively. 2. The results of antimicrobial susceptibility test revealed that the isolates showed high susceptibility to ciprofloxacin and cephalothin, moderate susceptibility to amikacin, gentamicin, kanamycin and streptomycin, and low susceptibility to erythromycin, tylosin and sulfadimethoxin. 3. The isolates were varied in pathogenicity to mice. Median lethal dose of LE9402(serovar 2) and LE9511(serovar 5) were $9.2{\times}10^7$ CFU and $2.8{\times}10^7$%CFU, respectively. Specific pneumonic lesions were observed from the infected mice with clinical signs. Bacteria recovery rate was high in the lung, but low In heart blood and tracheas. 4. Serovar 2 was found to be more pathogenic than serovar 5 in guinea pig. Mortality on guinea pigs inoculated with serovar 2($5.4{\times}10^8-5.4{\times}10^6$CFU) and serovar 5($2.8{\times}10^8-2.8{\times}10^6$ CFU) was 20~40% and 40~80%, respectively. A severe hemorrhagic lesions and focal pneumonic lesions were observed from dead guinea pigs. Bacteria recovery rate was relatively higher in the lung than that of other organs. 5. In the SDS-PAGE analysis, OMP-enriched fractions of both isolates and reference strains contain common peptide bands equivalent to molecular weight of 17, 27, 42, 52 and 95Kd. In addition to common peptide bands, the bands which are specific to each isolate were also observed. The profiles of Sephadex G25 fractions showed 3 major peaks. The common peptide bands which were observed by SDS-PAGE of the crude OMPs were found in the peaks 1 and 2. 6. The OMPs extracted from serovar 2(LE9402) and serovar 5(LE9511) provided high level of protection in mice(70~80%) and pigs(100%). All animals inoculated with OMPs were seroconverted, showing micro-agglutination titer of 640 to 1280.

• Journal of the Korean Society of Food Science and Nutrition
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• v.32 no.4
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• pp.519-525
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• 2003

The effect of chitosan on antimicrobial effect, shelf-life, antioxidation, color, moisture content and pH of pork loins stored at 1$0^{\circ}C$ was investigated. All concentration of 5 kDa chitosan had very weak antimicrobial effect in all concentration against spoilage bacteria in meat. 30 kDa and 120 kDa chitosan had very strong those effects above 0.1%. The pork dipped in 1% of 30 kDa and 120 kDa chitosans extended a shelf-life. In antioxidation, the dipped pork has shown remarkable effects in all concentrations of 30 kDa and 120 kDa chitosans. Also, the external redness of them was maintained stably. Their moisture contents were decreased slightly with the higher molecular weight and concentration during storage. The results suggested that properties of porks were improved with dipping in 1.0% of 30 kDa and 120 kDa chitosan.

• KSBB Journal
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• v.14 no.1
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• pp.9-16
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• 1999

Transforming growth factor $\beta$1(TGF-$\beta$1) has potentials to be used as a new therapeutic agent. However, studies with TGF-$\beta$ were hindered by its high cost. In this study, we developed an improved method to purify TGF-$\beta$1 from human platelets, for which four purification steps were used: platelet extraction, gel filtration, cation exchange chromatography, and reverse phase high performance liquid chromatography. After a final step of purification, a pure protein with a molecular weight of 25,000 corresponding to the commercially available TGF-$\beta$1 was obtained, which were confirmed by silver staining and Western blotting after SDS-polyacrylamide gel electrophoresis (SDS-PAGE). It was confirmed by the inhibitory effects of TGF-$\beta$1 on a mink lung epithelial cell line that the purified TGF-$\beta$1 had its biological activity, whose activity is slightly higher than that of the commercially available TGF-$\beta$1. About 3.7$\mug of the purified TGF-$\beta$1 was obtained from 10 units of concentrated human platelets, the final yield of which is about 21%.