• Journal of Microbiology and Biotechnology
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• v.6 no.2
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• pp.86-91
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• 1996

Thermostable $\beta$-mannanase from Bacillus sp. YA-14 was purified by acetone precipitation, CM-cellulose, Sephadex G-100 and hydroxyapatite column chromatography from culture supernatant. The final enzyme preparation appeared to be homogeneous on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). $\beta$-Mannanase appeared to be a monomeric protein with a molecular weight of 67, 000 daltons. The optimal pH and temperature of the enzyme reaction were pH 6.0 and $75^{\circ}C$ , respectively. The enzyme was stable at a pH range of 6.0 to 9.0 and at temperatures between 45 and $85^{\circ}C$. The kinetic constants of $\beta$-mannanase as determined with a galactomannan (locust bean) as substrate were a Vmax of 25 unit/ml and a Km of 1.1 mg/ml. The enzyme had only limited activity on galactomannan substrate. It was suggested that mg $\beta$-mannanase activity is limited by the number of branched $\alpha$-galactose residues.

• BMB Reports
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• v.35 no.2
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• pp.165-171
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• 2002

A serine collagenolytic protease was purified from the internal organs of filefish Novoden modestrus, by ammonium sulfate, ion-exchange chromatography on a DEAE-Sephadex A-50, ion-exchange rechromatography on a DEAE-Sephadex A-50, and gel filtration on a Sephadex G-150 column. The molecular mass of the filefish serine collagenase was estimated to be 27.0 kDa by gel filtration and SDS-PAGE. The purified collagenase was optimally active at pH 7.0-8.0 and $55^{\circ}C$. The purified enzyme was rich in Ala, Ser, Leu, and Ile, but poor in Trp, Pro, Tyr, and Met. In addition, the purified collagenolytic enzyme was strongly inhibited by N-P-toluenesulfonyl-L-lysine chloromethyl ketone (TLCK), diisopropylfluorophosphate (DFP), and soybean trypsin inhibitor.

• Korean Journal of Microbiology
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• v.31 no.3
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• pp.237-244
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• 1993

Aspartate aminotransferase (ASAT) (L-aspartate : 2-oxyoglutarate, EC 2.6. 1. 1.) from Streptomyces fradiae NRRL 2702 has been purified by acetone precipitation, DEAE-cellulose, hydroxyapatite, and preparative electrophoresis (Prep cell), of which the last was the most effective step in the purification of ASAT. The molecular mass was estimated to be 54,000 dalton by SDS-PAGE and 120,000 dalton by gel filtration chromatography. Preparative isoelectric focusing of purified ASAT resulted in one polypeptide band with a pI of 4.2, showing homogeneity and indicating that the enzyme is composed of two identical subunits. The enzyme was specific for L-aspartate as an amino donor ; the $K_{m}$ values were determined to be 2.7 mM for L-aspartate, 0.7 mM for 2-oxoglutarate, 12.8 mM for L-glutamate, and 0.15 mM for oxaloacetate. The enzyme was relatively heat-stable, having maximum activity at 55.deg.C, and it had a broad pH optimum ranging from 5.5 to 8.0. The activity of the purified enzyme was not inhibited by ammonium ions. This paper reports the first purification and characterization of the aspartate aminotransferase from a species of Streptomyces.s.

• 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.

• Journal of Life Science
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• v.8 no.3
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• pp.326-332
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• 1998

Cyclodextrin glucanotransferase from B. stearothermophilus KJ16 that can produce both cyclodextrin glucanotransferase and cyclodextrinase was purified by ammonium sulfate precipitation, DEAE-cellulose chromatography, Sephadex G-100 chromatography, and FPLC. The molecular weight of the purifice enzyme was about 65,000 dalton by SDS-PAGE. The optimal pH and temperature were 6.0 and $60^{\circ}C$, respectively. The enzyme was stable at $50^{\circ}C$ for 1 hr and in the pH range of 5.5 and 8.5. Mercaptoethanol and dithiothreitol inhibited the enzyme activity strongly. The enzyme produced 60% cyclodextrin(CD) from 5% soluble starch with the $^{\alpha}$, $^{\beta}$, $^{\gamma}$-CD ratio of 42:46:12. Amylopectin was the most suitable substrate with 67% conversion to CD.

• Journal of Microbiology
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• v.34 no.1
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• pp.82-89
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• 1996

Purine nucleoside phosphorylase (PNP) was purified in Micrococcus luteus (M. luteus) using streptomycin sulfate and amomonium sulfate fractionation, three times by a Sephadex G-100 gel filtration and a DEAE-Sephadex A-50 ion exchange chromatography. The enzyme was purified 72 folds with a 11% recovery and showed a single band in a nondenaturing gel electrophoresis. The M. W. of PNP turned out to be 1.35 * 10$^{5}$ delton in G-150 gel filtration chromatography. The stability of the enzyme was increased by treatment with both substrates, MgCI$_{2}$ or CaCI$_{2}$, but not significantly kcal/mol. M. luteus PNP catalyzed the phosphorolysis of inosine, deoxyinosine, guanosine and deoxyguanosine with the Km value of 1.5 * 10$^{-3}$ M, 3.0 * 10$^{-3}$ M, 5.0 * 10$^{-4}$ M, respectively. The enzyme was reacted with adenosine, 1-methylnosine and 1-methylguanosine as substrates, which were shown to be poor substrates for mammalian enzyme.

• Proceedings of the Zoological Society Korea Conference
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• 1997.10b
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• pp.257.1-257
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• 1997

No Abstract, See Full Text

• BMB Reports
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• v.41 no.3
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• pp.254-258
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• 2008

Lipase was purified from squid (Todarodes pacificus) liver in an attempt to investigate the possibility of applying the enzyme for biotechnological applications. Crude extract of squid liver was initially fractionated by the batch type ion exchange chromatography. The fraction containing lipase activity was further purified with an octyl-Sepharose column. Finally, lipase was purified by eluting active protein from a non-dissociating polyacrylamide gel after zymographic analysis. Molecular weight of the purified enzyme was determined to be 27 kDa by SDS-polyacrylamide gel electrophoresis. The enzyme showed the highest activity at a temperature range of $35-40^{\circ}C$ and at pH 8.0. The activity was almost completely inhibited at 1 mM concentration of $Hg^{2+}$ or $Cu^{2+}$ ion. Partial amino acid sequence of the enzyme was also determined.

• Journal of Microbiology and Biotechnology
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• v.14 no.4
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• pp.829-835
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• 2004

A fibrinolytic enzyme has been found in several bacteria isolated from fermented food. This study was carried out to investigate the purification and characteristics of the fibrinolytic enzyme produced by Bacillus subtilis KCK-7 originated from Chungkookjang. The fibrinolytic enzyme was purified to homogeneity from the culture supernatant using ammonium sulfate fractionation and chromatographies on DEAE-cellulose and on Sephadex G-100. The final specific activity of the purified enzyme increased 11.0-fold, and the protein amount in the purified enzyme was about 16% of that in the culture supernatant. The molecular weight of the purified enzyme was estimated to be about 45,000 by SDS-PAGE. The optimum pH and temperature for the enzyme activity were pH 7.0 and $60^{\circ}C$, respectively. The enzyme activity was relatively stable up to $60^{\circ}C$ over the pH range of 7.0-10.0. The fibrinolytic enzyme activity increased by $Ca^{2+}$ and $Cu^{2+}$, whereas it was inhibited by $Hg^{2+}$ and $Ba^{2+}$. In addition, it was severely inhibited by PMSF and DFT. It is suggested that the purified enzyme was a serine protease for the fibrinolysis. The purified enzyme could completely hydrolyze fibrin in vitro within 8 h. Hence, it is suggested that the purified enzyme can be put into practice as an effective thrombolytic agent.

• Journal of the Korean Society of Food Science and Nutrition
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• v.23 no.3
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• pp.490-495
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• 1994

Acid phosphatase (EC3.1.3.2) from carrots was partially purified by ammonium sulfate fractionation (30%-80%), Sephacryl S-200 gel filtration, cm-Sepharose CL-6B and DEAE -Sephacel ion exchange chromatography. The optimum ph and temperature of acid phosphatase from carrots were pH 5.5 and 55$^{\circ}C$, respectively. The enzyme was most stable at ph 6.0 and relatively unstable below pH 4.0 . The activation energy of the enayme was determined to be 10.6kcal/mole. The enzyme utilized p-nitrophenyl phosphate as a substrate among tested possible substrates, whereas it hydrolyzed 5' -IMP and 5'-GMP poorly. The Michaelis -Menten constant(Km) of the enzyme with p-nitrophenyl phosphate as a substrate was identified as 0.55mM. Amongtested metal ions and inhibitors, Al+++ Zn++, Cu++ , fluoride, metavanadate and molybdate ions inhibited the enzyme activity drastically.