• Journal of Microbiology and Biotechnology
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• v.20 no.4
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• pp.659-665
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• 2010

A cis-epoxysuccinate hydrolase (CESH) from Bordetella sp. BK-52 was purified 51.4-fold with a yield of 27.1% using ammonium sulfate precipitation, ionic exchange, hydrophobic interaction, molecular sieve chromatography and an additional anion-exchange chromatography. The CESH was stable in a broad range of temperature (up to $50^{\circ}C$) and pH (4.0-10.0) with optima of $40^{\circ}C$ and pH 6.5, respectively. It could be partially inhibited by EDTA-$Na_2$, $Ag^+$, SDS, and DTT, and slightly enhanced by $Ba^{2+}$ and $Ca^{2+}$. The enzyme exhibited high stereospecificity in D(-)-tartaric acid (enantiomeric excess value higher than 99%) with $K_m$ and $V_max$ values of 18.67 mM and $94.34\;{\mu}M$/min/mg for disodium cis-epoxysuccinate, respectively. The Bordetella sp. BK-52 CESH gene, which contained 885 nucleotides (open reading frame) encoding 294 amino acids with a molecular mass of about 32 kDa, was successfully overexpressed in Escherichia coli using a T7/lac promoter vector and the enzyme activity was increased 42-times compared with the original strain. It may be an industrial biocatalyst for the preparation of D(-)-tartaric acid.

• Journal of fish pathology
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• v.37 no.1
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• pp.79-88
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• 2024

Aeromonas spp. infections have been reported to cause significant economic losses not only in the ornamental fish industry but also in aquaculture. In December 2022-January 2023, an Aeromonas infection occurred on a goldfish in korea, A gram-negative bacterium was isolated from the skin and internal organs of infected goldfish (Carassius auratus). The results showed that the isolate was identified as Aeromonas veronii using 16S rDNA targeted oilgpnucleotide primers, furthermore characteristics of A. veronii was confirmed by enterotoxin gene, infectious experiment, antibiotic resistance. In-vivo pathogenicity of isolates to goldfsh resulted in 100% mortality in challenged host within one week of post experiment injection. As a result of PCR analysis targeting three enterotoxin-encoding genes, cytotoxic enterotoxin (act) was identified in A. veronii isolate in this study. Antimicrobial susceptibility pattern of isolate showed it was to susceptible to most antimicrobial agents tested but resistant to ampicillin, imipenem, meropenem and clindamycin.

• Proceedings of the Korean Society of Life Science Conference
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• 2001.06a
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• pp.67-86
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• 2001

A strain producing strongly fibrinolytic enzyme was isolated from soil and was identified to be Bacillus subtilis by biochemical and physiological characterization. The optimal culture conditions for the production of fibrinolytic enzyme was determined to be 1.0% tryptone, 1.5% soluble starch, 0.5% Peptone, 0.5% NaCl, $(NH_{4})_{3}PO_4.3H_{2}O, and MgSO_{4}.7H_{2}O.$ Initial pH and temperature were pH 8.0 and $30^{\circ}C$ , respectively, The highest enzyme production was observed at 30 hours of cultivation at $30^{\circ}C$ The fibrinolytic enzyme was purified to homogeneity by DEAE Sephadex A-50 ion exchange column chromatography, 70% ammonium sulfate precipitation, Sephadex G-200 and G-75 gel filtration column chromatography. The molecular weight of the purified enzyme was 28,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A gene encoding the fibrinolytic enzyme was cloned into a plasmid vector pBluescript, transforming E.coli XL-1 Blue. The clone was able to degrade fibrin, This indicated that the gene could encode a fibrinolytic enzyme. The nucleotide sequence of the 2.7 kb insert was determined in both direction. One open reading frame composed of 1023 nucleotides was found to be a potential protein coding region. There was the putative Shine-Dalgano sequence and TATA box upstream of the open reading frame. The homology search data in the genome database showed that both the 2.7 kb insert and 1 kb open reading frame carried no significance in the nucleotide sequence of known fibrinolytic enzyme from Bacillus serovars. The recombinant cell harboring the novel gene involved in fibrinolysis was subjected to protein purification. The molecular mass of the purified fibrinolytic enzyme was determined to be 31864 Dalton, which was highly in accordance with the molecular mass(33 kDa) of the fibrinolytic gene deduced from the insert. The fibrinolytic enzyme was Purified 50.5 folds to homogeneity in overall yield of 10.7% by DEAE Sephadex A-50 ion exchange, 85% ammonium sulfate precipitation, Sephadex G-50, Superdex 75 HR FPLC gel filtration. In conclusion, a novel fibrinolytic gene from Bacillus subtilis was identified and characterized by cloning a genomic library of Bacillus subtilis into pBleuscript. For the soybean fermented by this strain, it is found that there increased assistant protein about 20% compared to the soybean not fermented and increased about 30% according to amino acid analysis and, in particular, essential amino acid increased about 40%. When keeping this fermented soybean powder at room temperature for about 70days, it showed very high stability maintaining almost perfect activity and, therefore, it gave us great suggestion its possibility of development as a new functional food.

• 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.

• Korean Journal of Microbiology
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• v.45 no.4
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• pp.291-299
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• 2009

To elucidate HtrA3's functional roles in the HtrA3 mediated cellular processes, it is necessary to investigate its biochemical characteristics. In the present study, we constructed the plasmids encoding putative mature HtrA3 proteins (M1-HtrA3 and M2-HtrA3) based on the putative maturation sites of highly homologous HtrA1 and mouse HtrA3. We used the pGEX bacterial expression system to develop a simple and rapid purification for the recombinant HtrA3 protein. Although yields of the mature HtrA3 proteins were slightly low as 10~50 ${\mu}g$/L, the amounts and purity of M1- and M2-HtrA3 were enough to investigate their proteolytic activities. The putative mature HtrA3 proteins have proteolytic activity which could cleave $\beta$-casein as an exogenous substrate. We compared the proteolytic activity between the HtrA family, HtrA1, HtrA2, and HtrA3. The cleavage activity of HtrA3 and HtrA2 were 2 folds higher than that of HtrA1, respectively. Our study provides a method for generating useful reagents to identify natural substrates of HtrA3 in the further studies.

• Journal of Microbiology and Biotechnology
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• v.17 no.4
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• pp.638-643
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• 2007

The full encoding sequence for human type II hexokinase (HXK II) was cloned into the E. coli expression vector pET 21b and expressed as a C-terminally hexahistidine-tagged protein in the BL2l (DE3) strain. The IPTG-induced HXK II approximately accounted for 17% of the total E. coli proteins, and 81% of HXK $II_{6{\times}His}$ existed in inclusion bodies. To improve the production of soluble recombinant HXK II protein, in the functionally active form, we used low temperature, and the osmotic stress expression method. When expressed at $18^{\circ}C$, about 83% of HXK $II_{6{\times}His}$ existed in the soluble fraction, which amounted to a 4.1-fold yield over that expressed at $37^{\circ}C$. The soluble form of HXK $II_{6{\times}His}$ was also highly produced in the presence of 1M sorbitol under the standard condition $(37^{\circ}C)$, which indicated that temperature downshift and low water potentials were required to improve the yield of active recombinant HXK II protein. The expressed protein was purified by metal chelate affinity chromatography performed in an IDA Excellose column charged with $Ni^{2+}$ ions, resulting in about 40mg recombinant HXK II protein obtained with purity over 89% from 51 of E. coli culture. The identity of HXK $II_{6{\times}His}$ was confirmed by Western blotting analysis. Taken together, using the stress-governed expression described in this study, human active HXK II can be purified in sufficient amounts for biochemical and biomedical studies.

• Korean Journal of Microbiology
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• v.48 no.2
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• pp.163-170
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• 2012

In order to inhibit the growth of pathogens and degrade biogenic amines during the fermentation of soybean products, an isolate with antimicrobial activity against pathogens and biogenic amine-degrading property was obtained from 83 traditionally fermented soybean products. The morphological and biochemical tests and the phylogenetic relationship among 16S rRNA gene sequences indicated that the isolate named as the strain SCK B11 was most closely related to Bacillus licheniformis. The cell-free supernatant of two day cultures was active against several pathogens including Enterococcus faecalis, Listeria monocytosis, Micrococcus luteus, Pseudomonas aeruginosa, Bacillus cereus, and Staphylococcus aureus. PCR analysis was conducted to determine relatedness to antimicrobial lantibiotics and biosurfactants produced by Bacillus spp., but showed negative for the genes encoding surfactin, lichenysin, and lichenicidine. Electron microscopic observation indicated that the antimicrobial agent seemed to attack the membrane of the pathogens, leaving the ghost or shrunken cells. The strain was found to degrade histamine by 72% and tyramine by 66% in the cooked soybean containing 5.3% of biogenic amine over 10 days of fermentation time. The use of selected strain would be a potential control measure in manufacturing traditionally fermented soybean products that are difficult to control pathogens and biogenic amine levels.

• Toxicological Research
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• v.23 no.1
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• pp.55-63
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• 2007

Diethylnitrosamine (DEN) is a nitrosamine compound that can induce a variety of liver lesions including hepatic carcinoma, forming DNA-carcinogen adducts. In the present study, microarray analyses were performed with Affymetrix Murine Genome 430A Array in order to identify the gene-expression profiles for DEN and to provide valuable information for the evaluation of potential hepatotoxicity. C57BL/6NCrj mice were orally administered once with DEN at doses of 0, 3, 7 and 20 mg/kg. Liver from each animal was removed 2, 4, 8 and 24 hrs after the administration. The histopathological analysis and serum biochemical analysis showed no significant difference in DEN-treated groups compared to control group. Conversely, the principal component analysis (PCA) profiles demonstrated that a specific normal gene expression profile in control groups differed clearly from the expression profiles of DEN-treated groups. Within groups, a little variance was found between individuals. Student's t-test on the results obtained from triplicate hybridizations was performed to identify those genes with statistically significant changes in the expression. Statistical analysis revealed that 11 genes were significantly downregulated and 28 genes were upregulated in all three animals after 2 h treatment at 20 mg/kg. The upregulated group included genes encoding Gdf15, JunD1, and Mdm2, while the genes including Sox6, Shmt2, and SIc6a6 were largely down regulated. Hierarchical clustering of gene expression also allowed the identification of functionally related clusters that encode proteins related to metabolism, and MAPK signaling pathway. Taken together, this study suggests that match with a toxicant signature can assign a putative mechanism of action to the test compound if is established a database containing response patterns to various toxic compounds.

• Journal of Applied Biological Chemistry
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• v.54 no.2
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• pp.94-100
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• 2011

A gene encoding a putative phospholipase D was isolated from Bacillus licheniformis and cloned into pGEM-T easy vector. The gene was expressed in E. coli BL21 (DE3) using a pET-21(a) vector containing His6 tag. Affinity purification of the recombinant phospholipase D with nickel-nitrilotriacetic acid (Ni-NTA) resin resulted major one-band by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The purified enzyme showed a molecular weight of 44 kDa. The optimum activity of enzyme was around pH 7.0 and the enzyme was also the most stable around this condition. The optimum temperature was about $40-45^{\circ}C$ and the enzyme still showed considerable activities at wide range of temperature. Among various detergents, Triton X-100 significantly increased the enzyme activity, resulting in 181% activity of control at 0.6 mM of the detergent. Calcium ion did not significantly affect the enzyme activity, suggesting that the enzyme might be classified into $Ca^{2+}$-independent PLD.

• Journal of Applied Biological Chemistry
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• v.53 no.4
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• pp.195-201
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• 2010

A gene encoding an alanine racemase in B. pseudomycoides was cloned and one (Cys316) or both of two cysteines (Cys316 and Cys365) was (were) substituted with alanine. The cysteine (-) alanine racemases were expressed in E. coli BL21 (DE3) using a pET-21 vector. The expressed enzymes were purified through affinity chromatography using 6xHis ligand. The purified enzymes all showed major one bands by SDS-PAGE analysis, corresponding to 46 kDa. The cysteine (-) alanine racemases as well as the wild type enzyme showed alanine racemase activities, indicating that the enzyme is an alanine racemase and the cysteines in the enzyme may not be involved in the catalysis and/or substrate binding. Thermal stabilities of Cys (-) alanine racemases decreased considerably and half-lives were 26 (wild type), 21 (C316A) and 18 min (C316-365A), respectively at $60^{\circ}C$ pH 8.0, suggesting that cysteine is considerably contributive to the thermal stability of the alanine racemase.