• Parasites, Hosts and Diseases
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• 제41권4호
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• pp.189-196
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• 2003

In order to evaluate the possible roles of secretory proteases in the pathogenesis of amoebic keratitis, we purified and characterized a serine protease secreted by Acanthamoeba lugdunensis KA/E2, isolated from a Korean keratitis patient The ammonium sulfate-precipitated culture supernatant of the isolate was purified by sequential chromatography on CM-Sepharose, Sephacryl S-200, and mono Q-anion exchange column. The purified 33 kDa protease had a pH optimum of 8.5 and a temperature optimum of $55^{\circ}C$. Phenylmethylsulfonylfluoride and 4-(2-Aminoethyl)-benzenesulfonyl-fluoride, both serine protease specific inhibitors, inhibited almost completely the activity of the 33 kDa protease whereas other classes of inhibitors did not affect its activity. The 33 kDa enzyme degraded various extracellular matrix proteins and serum proteins. Our results strongly suggest that the 33 kDa serine protease secreted from this keratopathogenic Acanthamoeba play important roles in the pathogenesis of amoebic keratitis, such as in corneal tissue invasion, immune evasion and nutrient uptake.

• Journal of Microbiology and Biotechnology
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• 제29권7호
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• pp.1033-1042
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• 2019

Bacillus velezensis BS2 was isolated from meongge (common sea squirt) jeotgal, a Korean fermented seafood, and produces a bacteriocin, BacBS2, which strongly inhibits Listeria monocytogenes and Bacillus cereus. BacBS2 was partially purified by Q-Sepharose column chromatography after ammonium sulfate precipitation of the culture supernatant, then further purified by Sephadex G-50 column chromatography. Partially purified BacBS2 was estimated to be 6.5 kDa in size by Tricine-SDS PAGE and activity detection by gel-overlay. Enzyme treatment and FT-IR spectrum of partially purified BacBS2 confirmed its proteinaceous nature. BacBS2 was fully stable at pH 4-9, and half of activity was retained at pH 1-3. Full activity was retained after exposure to $80^{\circ}C$ for 15 min, but half of the activity was retained upon exposure to $90^{\circ}C$ for 15 min or $100^{\circ}C$for 10 min. BacBS2 inhibited L. monocytogenes by bactericidal mode of action. B. velezensis BS2 and its BacBS2 seem useful as biopreservatives for fermented foods such as jeotgal.

• Journal of Microbiology and Biotechnology
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• 제24권4호
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• pp.489-496
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• 2014

High levels of extracellular xylanase activity (211.79 IU/mg) produced by Paenibacillus sp. NF1 were detected when it was submerged-cultured. After three consecutive purification steps using Octyl-Sepharose, Sephadex G75, and Q-Sepharose columns, a thermostable xylanase (XynNF) was purified to homogeneity and showed a molecular mass of 37 kDa according to SDS-PAGE. The specific activity of the purified XynNF was up to 3,081.05 IU/mg with a 14.55-fold purification. The activity of XynNF was stimulated by $Ca^{2+}$, $Ba^{2+}$, DTT, and ${\beta}$-mercaptoethanol, but was inhibited by $Fe^{2+}$, $Zn^{2+}$, $Fe^{2+}$, $Cu^{2+}$, SDS, and EDTA. The purified XynNF displayed a greater affinity for oat spelt xylan with the maximal enzymatic activity at $60^{\circ}C$ and pH 6.0. XynNF, which was shown to be cellulose-free, with high stability at high temperature ($70^{\circ}C-80^{\circ}C$) and low pH range (pH 4.0-7.0), is potentially valuable for various industrial applications. The enzyme hydrolyzed oat spelt xylan to yield mainly xylooligosaccharides (95.8%) of 2-4 degree of polymerization (DP2-4). Moreover, the majority of the xylooligosacharides (DP2-4) products was xylobiose (61.5%). The thermostable xylanase (XynNF) thus seems potentially usefull in the production of xylooligosaccharides.

• Journal of Microbiology
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• 제41권3호
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• pp.207-211
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• 2003

Extracellular protease, from Aeromonas hydrophila Ni 39, was purified 16.7-fold to electrophoretic homogeneity with an overall yield of 19.9％, through a purification procedure of acetone precipitation, and Q Sepharose and Sephacryl S-200 chromatographies. The isoelectric point of the enzyme was 6.0 and the molecular mass, as determined by Sephacryl S-200 HR chromatography, was found to be about 102 kDa. SDS/PAGE revealed that the enzyme consisted of two subunits, with molecular masses of 65.9 kDa. Under standard assay conditions, the apparent $K_{m}$ value of the enzyme toward casein was 0.32 mg/ml. About 90％ of the proteolytic activity remained after heating at 60$^{\circ}C$ for 30 min. The highest rate of azocasein hydrolysis for the enzyme was reached at 60$^{\circ}C$, and the optimum pH of the enzyme was 9.0. The enzyme was inhibited by the serine protease inhibitor, phenylmethylsulfonyl fluoride (PMSF), by about 87.9％, but not by E64, EDTA, pepstatin or 1,10-phenanthroline. The enzyme activity was inhibited slightly by Ca$\^$2＋/, Mg$\^$2＋/ and Zn/supb 2＋/ ions.

• Journal of Microbiology and Biotechnology
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• 제18권3호
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• pp.457-464
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• 2008

An extracellular enzyme (RMEBE) possessing ${\alpha}-(1{\rightarrow}4)-(1{\rightarrow}6)$-transferring activity was purified to homogeneity from Rhodothermus marin us by combination of ammonium sulfate precipitation, Q-Sepharose ion-exchange, and Superdex-200 gel filtration chromatographies, and preparative native polyacrylamide gel electrophoresis. The purified enzyme had an optimum pH of 6.0 and was highly thermostable with a maximal activity at $80^{\circ}C$. Its half-life was determined to be 73.7 and 16.7 min at 80 and $85^{\circ}C$, respectively. The enzyme was also halophilic and highly halotolerant up to about 2M NaCl, with a maximal activity at 0.5M. The substrate specificity of RMEBE suggested that it possesses partial characteristics of both glucan branching enzyme and neopullulanase. RMEBE clearly produced branched glucans from amylose, with partial ${\alpha}-(1{\rightarrow}4)$-hydrolysis of amylose and starch. At the same time, it hydrolyzed pullulan partly to panose, and exhibited ${\alpha}-(1{\rightarrow}4)-(1{\rightarrow}6)$-transferase activity for small maltooligosaccharides, producing disproportionated ${\alpha}-(1{\rightarrow}6)$-branched maltooligosaccharides. The enzyme preferred maltopentaose and maltohexaose to smaller maltooligosaccharides for production of longer branched products. Thus, the results suggest that RMEBE might be applied for production of branched oligosaccharides from small maltodextrins at high temperature or even at high salinity.

• BMB Reports
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• 제32권3호
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• pp.239-246
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• 1999

A tripeptidase (PepT) was purified to homogeneity from Bacillus subtilis through four sequential chromatographies including DEAE-Sepharose ion exchange, hydroxylapatite, mono-Q FPLC ion exchange, and Superose-12 FPLC gel filtration. The apparent molecular mass of the enzyme was 49,200 Da and 51,400 Da as determined by sodium dodecylsulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration chromatography, respectively, and the enzyme exists in a monomeric form. The physicochemical properties of the enzyme were as follows: optimum pH at 7.5, optimum temperature at $60^{\circ}C$, and pI at 4.9. The $K_m$ and $V_{max}$ values of the enzyme were 4.3 mM and 2.5 mmol/min/mg, respectively, with MetAla-Ser as substrate. The B. subtilis PepT requires $Co^{2+}$ ion(s) for activation, while it is inactivated by EOTA and 1,10-phenanthroline, suggesting that it is a metalloprotein. The enzyme was not inhibited by any of serine protease, aspartic protease, or leucine aminopeptidase inhibitors. The enzyme showed comparable activities towards four different substrates including Met-Ala-Ser, Leu-Gly-Gly, Leu-Ser-Phe, and Leu-Leu-Tyr. The amino terminal sequence of PepT determined by Edman degradation was found to be MKEEIIERFTTYVXV and turned out to be identical to that of PepT deduced from a cloned B. subtilis pepT.

• Journal of Microbiology and Biotechnology
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• 제11권6호
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• pp.1011-1017
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• 2001

Streptomyces sp. Y-110 cytochrome P-450, induced by the addition of compactin -Na into the culture medium, was purified from the cell extract to apparent homogeniety, mainly by DEAE-Sepharose, hydroxyapatite, and Mono Q column chromatyography. The sepcific activity of purified enzyme on its substrate, compactin-Na, was determined to be 15 nmol of pravastatin per mg protein. The molecular mass of this enzyme on SDS-PAGE was $37{\pm}0.5$ kDa, pI was 4.5, and its CO difference spectrum showed maximum absorption peaks at 452 and 550nm, respectively. The N-terminal amino acid sequence was determined to be Met>Thr>Cys>Thr>Pro>Val>Thr>Val>The>Gly>Ala>Ala>Gly>Gln>Ile>Gly>Tyr>Ala>Leu. Its apparent $K_m$ on compactin-Na was $1.294{\mu}M{\cdot}min^-1,\;and\;V_{max}\;was\;1.028{\mu}M{\cdot}min^-1$. The maximum substrate concentration ($K_s$) for reaction was $270 {\mu}M$and thus $1/[K_s]$ was $3.7{\mu}M$. These physicochemical characteristics and kinetic behavior of this enzyme were compared and shown to be different from those of Streptomyces cytochrome P-450 enzymes reported, suggesting that this enzyme may be an additional member of the Streptomyces cytochrome P-450 family.

• Journal of Microbiology and Biotechnology
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• 제25권9호
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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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• 제12권6호
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• pp.972-978
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• 2002

Streptomyces sp. AD001 is a Gram-positive soil actinomycetes secreting an uncharacterized 2,4-dichlorophenol (DCP) oxidizing enzyme, whose activity is similar to the previously known Actinomycetes lignin-peroxidase (ALiP). This extracellular peroxidase was purified from Streptomyces sp. AD001 as a single protein band on an SDS-PACE by ammonium sulfate fractionation, Q-sepharose, concanavalin A, and Bio-Gel HTP column chromatographies. The molecular mass of the purified peroxidase was determined by SDS-PAGE to be 45.2 kDa, and 49.7 kDa with MALDI-TOF-MS, respectively. The highest level of peroxidase activity was observed at pH 7.5 and $30^{\circ}C$. The amino terminal sequence of the purified peroxidase (G-E-P-E-E-G-N-V-D-G-T-L) showed no significant homologies to my known proteins, suggesting that Streptomyces sp. AD001 may secrete a novel kind of bacterial peroxidase Initial rate kinetic data of the 2,4-DCP oxidation were best modeled with a random-binding bireactant system.

• Journal of Microbiology and Biotechnology
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• 제20권7호
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• pp.1069-1076
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• 2010

A novel laccase from Tricholoma mongolicum was purified by using a procedure that entailed ion-exchange chromatographies on DEAE-cellulose, CM-cellulose, and Q-Sepharose, and FPLC-gel filtration on Superdex 75. The purified enzyme was obtained with a specific activity of 1,480 U/mg-protein and a final yield of 15%. It was found to be a monomeric protein with a molecular mass of 66 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its N-terminal amino acid sequence was GIGPVADLYVGNRIL, similar to some but also different to other mushroom laccases. The optimum pH and temperature for the purified enzyme were pH 2 to pH 3 and $30^{\circ}C$, respectively. It displayed a low $K_m$ toward 2,7-azinobis (3-ethylbenzothiazolone-6-sulfonic acid) diammonium salt (ABTS) and high $k_{cat}/K_m$ values. The purified laccase oxidized a wide range of lignin-related phenols, but exerted maximal activity on ABTS. It was significantly inhibited by $Hg^{2+}$ ions, and remarkably stimulated by $Cu^{2+}$ ions. It inhibited HIV-1 reverse transcriptase and proliferation of hepatoma HepG2 cells and breast cancer MCF7 cells with an $IC_{50}$ of 0.65 ${\mu}M$, 1.4 ${\mu}M$, and 4.2 ${\mu}M$, respectively, indicating that it is also an antipathogenic protein.