• Journal of Microbiology and Biotechnology
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• v.20 no.4
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• pp.789-797
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• 2010

The limited information on differential gene expression in the different serotypes of Actinobacillus pleuropneumoniae has significantly hampered the research on the pathogenic mechanisms of this organism and the development of multivalent vaccines against A. pleuropneumoniae infection. To compare the gene expressions in the A. pleuropneumoniae strains CVCC259 (serotype 1) and CVCC261 (serotype 3), we screened the differentially expressed genes in the two strains by performing representational difference analysis (RDA). Northern blot analyses were used to confirm the results of RDA. We identified 22 differentially expressed genes in the CVCC259 strain and 20 differentially expressed genes in the CVCC261 strain, and these genes were classified into 11 groups: (1) genes encoding APX toxins; (2) genes encoding transferrin-binding protein; (3) genes involved in lipopolysaccharide (LPS) biosynthesis; (4) genes encoding autotransporter adhesin; (5) genes involved in metabolism; (6) genes involved in the ATP-binding cassette (ABC) transporter system; (7) genes encoding molecular chaperones; (8) genes involved in bacterial transcription and nucleic acid metabolism; (9) a gene encoding protease; (10) genes encoding lipoprotein/membrane protein; and (11) genes encoding various hypothetical proteins. This is the first report on the systematic application of RDA for the analysis of differential gene expression in A. pleuropneumoniae serotypes 1 and 3. The determination of these differentially expressed genes will serve as an indicator for future research on the pathogenic mechanisms of A. pleuropneumoniae and the development of a multivalent vaccine against A. pleuropneumoniae infection.

• Journal of Microbiology and Biotechnology
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• v.24 no.2
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• pp.245-253
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• 2014

A fibrinolytic enzyme was produced by an edible mushroom of Pleurotus ostreatus using submerged culture fermentation. The enzyme was purified from the culture supernatant by applying a combination of freeze-thaw treatment, ammonium sulfate precipitation, hydrophobic interaction, and gel filtration chromatographies. The enzyme was purified by a 147-fold, with a yield of 7.54%. The molecular masses of the enzyme an determined by gel filtration and SDS-PAGE were 13.6 and 18.2 kDa, respectively. The isoelectric point of the enzyme was 8.52. It hydrolyzed fibrinogen by cleaving the ${\alpha}$ and ${\beta}$ chains of fibrinogen followed by the ${\gamma}$ chains, and also activated plasminogen into plasmin. The enzyme was optimally active at $45^{\circ}C$ and pH 7.4. The enzyme activity was completely inhibited by EDTA, whereas protease inhibitors of TPCK, SBTI, PMSF, aprotinin and pepstatin showed no inhibition on its activity. The partial amino acid sequences of the enzyme as determined by Q-TOF2 were ATFVGCSATR, GGTLIHESSHFTR, and YTTWFGTFVTSR. These sequences showed a high degree of homology with those of metallo-endopeptidases from P. ostreatus and Armillaria mellea. The purified enzyme can also be applied as a natural agent for oral fibrinolytic therapy or prevention of thrombosis.

• Microbiology and Biotechnology Letters
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• v.46 no.1
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• pp.1-8
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• 2018

Gul (oyster) jeotgals (GJs) were prepared using different types of salt (23%, w/v): purified salt, solar salt aged for 3 years, and bamboo salt crystalized 3 times. One set of GJs was fermented with Bacillus subtilis JS2 ($10^6CFU/g$), while the other GJ set was fermented without starter. During fermentation for 24 weeks at $15^{\circ}C$, the starter GJs showed 10-fold higher bacilli counts than the no-starter GJs, where the maximum bacilli count was $8{\times}10^3CFU/g$. All 28 bacilli strains isolated from the 6-week GJs were identified as B. subtilis by using a RAPD-PCR, indicating that some of the B. subtilis JS2 cells remained viable. Lactic acid bacteria (LAB) and yeasts were present at low levels, $10^1-10^2CFU/g$. LAB with protease activities isolated from 10-week samples were identified as Enterococcus species. The isolates obtained at 16 weeks were all Staphylococcus species. The GJs with bamboo salt showed higher pH and lower titratable acidity (TA) values than the other GJs due to the strong alkalinity of bamboo salt. The amino-type nitrogen in the GJs increased slowly during the fermentation. At 24 weeks, the GJs with purified salt showed the highest amino-type nitrogen (412-430 mg%), followed by the GJs with solar salt (397-406 mg%) and GJs with bamboo salt (264-276 mg%). Meanwhile, the GJs with bamboo salt showed the highest ammonia-type N (63.67 mg%), followed by the GJs with purified salt (49 mg%) and solar salt (48 mg%).

• Journal of Microbiology and Biotechnology
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• v.10 no.5
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• pp.612-619
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• 2000

To investigate the biochemical properties of V. mimicus metalloprotease, whose gene was isolated previously from Vibrio mimicus ATCC33653, overexpression and purification were attempted. The 1.9 kb of open reading frame was amplified by PCR from pVMC193 plasmid which ligated the VMC gene with pUC19 and introduced into Escherichia coli BL21 (DE3) using the overexpression vector, pET22b (+). The overexpressed metalloprotease (VMC) was purified with Ni-NTA column chromatography and characterized with various protease inhibitors, pHs, temperatures, and substrates. The purified VMC showed the proteolytic activity against gelatin, soluble and insoluble collagens, and synthetic peptides. Unlike the observations made with all metalloproteases originated from other Vibrio sp., the VMC did not hydrolyze the casein. The proteolytic activity was critically decreased when the VMC was treated with metal chelating reagents, such as EDTA, 2,2-bipyridine, and 1, 10-phenanthroline. In particular, the 71 kDa VMC exhibited the hemagglutinating activity against human erythrocyte. As the purified VMC was treated with $CuCl_2$ and $NiCl_2$ for the chemical modification of metal binding, the proteolytic activity and hemagglutinating activity were profoundly influenced. The multialignment analysis made on the reported Vibrio metalloproteases showed the difference of amino acid sequence similarity between the two distinctive classes of Vibrio metalloproteases.

• Journal of Microbiology and Biotechnology
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• v.25 no.9
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• pp.1493-1501
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• 2015

A Bacillus species, EMD4, with strong antibacterial activity was isolated from ganjang (soy sauce) and identified as B. subtilis. B. subtilis EMD4 strongly inhibited the growth of B. cereus ATCC14579 and B. thuringiensis ATCC33679. The antibacterial activity was stable at pH 3-9 but inactive at pH 10 and above. The activity was fully retained after 15 min at 80℃ but reduced by 50% after 15 min at 90℃. The activity was completely destroyed by proteinase K and protease treatment, indicating its proteinaceous nature. The bacteriocin (BacEMD4) was partially purified from culture supernatant by ammonium sulfate precipitation, and Q-Sepharose and Sephadex G-50 column chromatographies. The specific activity was increased from 769.2 AU/mg protein to 8,347.8 AU/mg protein and the final yield was 12.6%. The size of BacEMD4 was determined to be 3.5 kDa by Tricine SDS-PAGE. The N-terminal amino acid sequence was similar with that of Subtilosin A. Nucleotide sequencing of the cloned gene confirmed that BacEMD4 was Subtilosin A. BacEMD4 showed bactericidal activity against B. cereus ATCC14579.

• Journal of Microbiology and Biotechnology
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• v.28 no.9
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• pp.1482-1492
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• 2018

Fusarium oxysporum trypsin (FOT) is a fungal serine protease similar to mammal trypsin. The FOT could be successfully expressed in Pichiapastoris by engineering the natural propeptide APQEIPN. In this study, we constructed two recombinant enzymes with engineered amino acid sequences added to the N-terminus of FOT and expressed in P. pastoris. The N-terminal residues had various effects on the structural and functional properties of trypsin. The FOT, and the recombinants TE (with peptide YVEF) and TS (with peptide YV) displayed the same optimum temperature ($40^{\circ}C$) and pH (8.0). However, the combinants TE and TS showed significantly increased thermal stability at $40^{\circ}C$ and $50^{\circ}C$. Moreover, the combinants TE and TS also showed enhanced tolerance of alkaline pH conditions. Compared with those of wild-type FOT, the intramolecular hydrogen bonds and the cation ${\pi}$-interactions of the recombinants TE and TS were significantly increased. The recombinants TE and TS also had significantly increased catalytic efficiencies (referring to the specificity constant, $k_{cat}/K_m$), 1.75-fold and 1.23-fold than wild-type FOT. In silico modeling analysis uncovered that the introduction of the peptides YVEF and YV resulted in shorter distances between the substrate binding pocket (D174, G198, and G208) and catalytic triad (His42, Asp102, and Ser180), which would improve the electron transfer rate and catalytic efficiency. In addition, N-terminal residues modification described here may be a useful approach for improving the catalytic efficiencies and characteristics of other target enzymes.

• Journal of Microbiology and Biotechnology
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• v.26 no.4
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• pp.666-674
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• 2016

The bacterial strains were screened as potential starters for fermenting low-salt doenjang (a Korean traditional fermented soybean paste) using Korean doenjang based on proteolytic and antipathogenic activities under 6.5-7.5% NaCl conditions. Phylogenetic analysis based on 16S rRNA gene sequences showed that they all belonged to the genus Bacillus. Proteolytic and antipathogenic activities against Escherichia coli, Bacillus cereus, Staphylococcus aureus, Listeria monocytogenes, and Aspergillus flavus, as well as fibrinolytic, amylase, and cellulase activities of the 10 strains were quantitatively evaluated. Of these, strains D2-2, JJ-D34, and D12-5 were selected, based on their activities. The functional, phenotypic, and safety-related characteristics of these three strains were additionally investigated and strains D2-2 and D12-5, which lacked antibiotic resistance, were finally selected. Strains D2-2 and D12-5 produced poly-γ-glutamic acid and showed various enzyme activities, including α-glucosidase and β-glucosidase. Growth properties of strains D2-2 and D12-5 included wide temperature and pH ranges, growth in up to 16% NaCl, and weak anaerobic growth, suggesting that they facilitate low-salt doenjang fermentation. Strains D2-2 and D12-5 were not hemolytic, carried no toxin genes, and did not produce biogenic amines. These results suggest that strains D2-2 and D12-5 can serve as appropriate starter cultures for fermenting low-salt doenjang with high quality and safety.

• The KIPS Transactions:PartD
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• v.10D no.2
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• pp.261-266
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• 2003

A protein, which is a linear polymer of amino acids, is one of the most important bio-molecules composing biological structures and regulating bio-chemical reactions. Since the characteristics and functions of proteins are determined by their amino acid sequences in principle, protein sequence determination is the starting point of protein function study. This paper proposes a protein sequence prediction method based on data mining techniques, which can overcome the limitation of previous bio-chemical sequencing methods. After applying multiple proteases to acquire overlapped protein fragments, we can identify candidate fragment sequences by comparing fragment mass values with peptide databases. We propose a method to construct multi-partite graph and search maximal paths to determine the protein sequence by assembling proper candidate sequences. In addition, experimental results based on the SWISS-PROT database showing the validity of the proposed method is presented.

• Fisheries and Aquatic Sciences
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• v.18 no.1
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• pp.13-20
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• 2015

Calculated chemical scores (computed in relation to the FAO/WHO reference protein) for salmon liver protein hydrolysates indicated that all amino acids (other than methionine and threonine) were present in adequate or excess quantities; thus, the raw liver material is a good source of essential amino acids. The hydrophobic amino acids contents in hydrolysates prepared from Oncorhynchus keta and O. gorbuscha were 38.4 and 39.1%, respectively. The proportion of released peptides exceeding 500 kDa was reduced when hydrolysates were treated with the commercial enzyme Alcalase, although proportions in the following MW ranges were elevated: 100-500 kDa and <50 kDa. The optimal conditions for enzymatic hydrolysis were as follows: pH 7.0, $50^{\circ}C$, and a reaction time of 1 h. Of the different proteases tested, Alcalase was the most efficient for production of salmon liver hydrolysate with the highest 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging activity. The hydrolysates prepared from salmon liver had a balanced amino acid composition. The liver protein hydrolysates contained low molecular weight peptides, some of which may be bio-active; this bio-active potential should be investigated. Inhibition of the DPPH radical increased with increased degree of hydrolysis (DH), regardless of protease type. DPPH radical scavenging abilities, antithrombotic effects and ${\alpha}$-glucosidase enzyme inhibition effects of O. keta liver hydrolysate increased in a dose-dependent manner. Thus, salmon liver hydrolysate may be useful in functional food applications and as a source of novel products.

• Parasites, Hosts and Diseases
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• v.42 no.2
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• pp.61-66
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• 2004

The plasmepsins are the aspartic proteases of malaria parasites. Treatment of aspartic protease inhibitor inhibits hemoglobin hydrolysis and blocks the parasite development in vitro suggesting that these proteases might be exploited their potentials as antimalarial drug targets. In this study, we determined the genetic variations of the aspartic proteases of Plasmodium vivax (PvPMs) of wild isolates. Two plasmepsins (PvPM4 and PvPM5) were cloned and sequenced from 20 P. vivax Korean isolates and two imported isolates. The sequences of the enzymes were highly conserved except a small number of amino acid substitutions did not modify key residues for the function or the structure of the enzymes. The high sequence conservations between the plasmepsins from the isolates support the notion that the enzymes could be reliable targets for new antimalarial chemotherapeutics.