• Journal of Microbiology
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• v.44 no.2
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• pp.185-191
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• 2006

The photosynthetic bacterium, Rhodospirillum rubrum S1, when grown under anaerobic conditions, generated three different types of catalases. In this study, we purified and characterized the highest molecular weight catalase from the three catalases. The total specific catalase activity of the crude cell extracts was 88 U/mg. After the completion of the final purification step, the specific activity of the purified catalase was 1,256 U/mg. The purified catalase evidenced an estimated molecular mass of 318 kDa, consisting of four identical subunits, each of 79 kDa. The purified enzyme exhibited an apparent Km value of 30.4 mM and a Vmax of 2,564 U against hydrogen peroxide. The enzyme also exhibited a broad optimal pH $(5.0{\sim}9.0)$, and remained stable over a broad temperature range $(20^{\circ}C{\sim}60^{\circ}C)$. It maintained 90% activity against organic solvents (ethanol/chloroform) known hydroperoxidase inhibitors, and exhibited no detectable peroxidase activity. The catalase activity of the purified enzyme was reduced to 19 % of full activity as the result of the administration of 10 mM 3-amino-1,2,4-triazole, a heme-containing catalase inhibitor. Sodium cyanide, sodium azide, and hydroxylamine, all of which are known heme protein inhibitors, inhibited catalase activity by 50 % at concentrations of $11.5{\mu}M,\;0.52{\mu}M,\;and\;0.11{\mu}M$, respectively. In accordance with these findings, the enzyme was identified as a type of monofunctional catalase.

• Journal of Microbiology and Biotechnology
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• v.17 no.5
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• pp.745-752
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• 2007

A $\beta$-glucosidase from the algal lytic bacterium Sinorhizobium kostiense AFK-13, grown in complex media containing cellobiose, was purified to homogeneity by successive ammonium sulfate precipitation, and anion-exchange and gel-filtration chromatographies. The enzyme was shown to be a monomeric protein with an apparent molecular mass of 52 kDa and isoelectric point of approximately 5.4. It was optimally active at pH 6.0 and $40^{\circ}C$ and possessed a specific activity of 260.4 U/mg of protein against $4-nitrophenyl-\beta-D-glucopyranoside$(pNPG). A temperature-stability analysis demonstrated that the enzyme was unstable at $50^{\circ}C$ and above. The enzyme did not require divalent cations for activity, and its activity was significantly suppressed by $Hg^{+2}\;and\;Ag^+$, whereas sodium dodecyl sulfate(SDS) and Triton X-100 moderately inhibited the enzyme to under 70% of its initial activity. In an algal lytic activity analysis, the growth of cyanobacteria, such as Anabaena flos-aquae, A. cylindrica, A. macrospora, Oscillatoria sancta, and Microcystis aeruginosa, was strongly inhibited by a treatment of 20 ppm/disc or 30 ppm/disc concentration of the enzyme.

• Applied Biological Chemistry
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• v.46 no.3
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• pp.176-182
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• 2003

An alkaline fibrinolytic protease-producing bacteria was isolated front Korean traditional soy sauce and identified as Bacillus subtilis K7 from the results of analyses of its morphological and physiological properties, $API^{\circledR}$, and Biolog system. The enzyme was purified by 75% ammonium sulfate fractionation, QAE-Sephadex anion and SP-Sephadex cation exchange column chromatography and Sephadex G-100 gel filtration. The specific activity of the purified enByme was 233.9 unit/mg protein and the yield of enzyme was 3.8%. The homogeneity of the purified enzyme was confirmed by polyacrylamide gel electrophoresis. Molecular mass of the enzyme was estimated about 21,500 Da by SDS-polyacrylamide get electrophoresis and gel chromatography. The optimum temperature and pH for the enzyme activity were $40^{\circ}C$ and 9.0, respectively. The enzyme was stable in a pH range of 5.0 to 12.0, and 60% of its activity was lost on heat treatment at $50^{\circ}C$ for 20 min. The activity of the purified enzyme was inhibited by the presence of $Fe^{2+},\;Ag^{2+},\;Cu6{2+}$, iodoacetate, ethylene diamine tetraacetic acid (EDTA), and trans-1,2-diaminocycloheane-N,N,N',N'-tetraacetic acid (CDTA). The results indicates that the enzyme requires a metal ion for its enzymatic activity.

• Food Science and Biotechnology
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• v.17 no.4
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• pp.766-771
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• 2008

The proteolytic enzyme from Saccharomyces sp. B101 was purified to homogeneity by ammonium sulfate fractionation, ultrafiltration, diethyl aminoethyl (DEAE)-Sephadex A-50 ion-exchange chromatography, and Sephadex G-100 gel filtration chromatography from the culture supernatant of Saccharomyces sp. B101. The specific activity and the purification fold of the purified enzyme were 4,688.9 unit/mg and 18, respectively. The molecular weight of the purified enzyme was estimated to be 33 kDa by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH and temperature for the enzyme activity were pH 8.5 and $30^{\circ}C$, respectively. The enzyme activity was relatively stable in the pH range of 6.5-8.5 at below $35^{\circ}C$. The salt-tolerance and stability for the enzyme activity were relatively stable even at NaCl concentrations of 10 and 15%. The activity of enzyme was inhibited by $Ag^{2+}$ and $Fe^{2+}$, and activated by $Mn^{2+}$. In addition, the enzyme activity was potently inhibited by ethylenediaminetetraacetic acid (EDTA) and phenylmethyl sulfonylfluoride (PMSF). Based on these findings we concluded that the purified enzyme was a serine protease. Km and Vmax values for hammastein milk casein were 1.02 mg/mL and 278.38 unit/mL, respectively.

• BMB Reports
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• v.29 no.4
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• pp.321-326
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• 1996

Spinach glycolate oxidase was subjected to a series of chemical modifications aimed at identifying amino acid residues essential for catalytic activity. The oxidase was reversibly inactivated by treatment with pyridoxal 5'-phosphate (PLP). The inactivation by PLP was accompanied by the appearance of an absorption peak of around 430 nm, which was shifted to 325 nm upon reduction with $NaBH_4$. After reduction, the PLP-treated oxidase showed a fluorescence spectrum with a maximum of around 395 nm by exciting at 325 nm. The substrate-competitive inhibitors oxalate and oxaloacetate provided protection against inactivation of the oxidase by PLP. These results suggest that PLP inactivates the enzyme by fonning a Schiff base with lysyl residue(s) at an active site of the oxidase. The enzyme was also inactivated by tryptophan-specific reagent N-bromosuccinimide (NBS). However, competitive inhibitors oxalate and oxaloacetate could not protect the oxidase significantly against inactivation of the enzyme by NBS. The results implicate that the inactivation of the oxidase by NBS is not directly related to modification of the tryptophanyl residue at an active site of the enzyme. Treatments of the oxidase with cysteine-specific reagents iodoacetate, silver nitrate, and 5,5'-dithiobis-2-nitrobenzoic acid did not affect significantly the activity of the enzyme.

• 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.

• BMB Reports
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• v.37 no.4
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• pp.387-393
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• 2004

The L-asparaginase (E. C. 3. 5. 1. 1) enzyme was purified to homogeneity from Pseudomonas aeruginosa 50071 cells that were grown on solid-state fermentation. Different purification steps (including ammonium sulfate fractionation followed by separation on Sephadex G-100 gel filtration and CM-Sephadex C50) were applied to the crude culture filtrate to obtain a pure enzyme preparation. The enzyme was purified 106-fold and showed a final specific activity of 1900 IU/mg with a 43% yield. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified enzyme revealed it was one peptide chain with $M_r$ of 160 kDa. A Lineweaver-Burk analysis showed a $K_m$ value of 0.147 mM and $V_{max}$ of 35.7 IU. The enzyme showed maximum activity at pH 9 when incubated at $37^{\circ}C$ for 30 min. The amino acid composition of the purified enzyme was also determined.

• The Korean Journal of Food And Nutrition
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• v.11 no.5
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• pp.576-579
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• 1998

Water extract of mulberry tree barks(Morus alba Linne) was studied for its proteolytic activity. Protein content of the extract was 1.12mg/ml and its specific activity was 5.14U/ml. The enzyme was active on various proteins : the relative acitities were 100 for casein, 63 for albumin, 58 for collagen, 45 for hemoglobin and 36 gelatin, respectively. There suggested that the ability of the enzyme to hydrolyze meat was relatively high since those are major meat proteins. Optimum pH and temperature for proteolytic activity were : pH 6.0 and 6$0^{\circ}C$. And the enzyme was stable at the pH range of 6.0 to 7.0 and temperature between 50 and 8$0^{\circ}C$. Apparent proteolytic activities could support some scientific grounds of traditional application of mulberry tree barks to home cooking for meat tenderization.

• Parasites, Hosts and Diseases
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• v.29 no.3
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• pp.259-266
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• 1991

In cytosolic (raction of adult Paragonimus westermani, superoxide dismutase activity was identified (4.3 units/mg of specific activity) using a xanthine-xanthine oxidase system. The enzyme was purified 150 fold in its activity using the ammonium sulfate precipitation, DEAE-Trisacryl M anion-exchange chromatography and Sephadex G-100 molecular sieve chromatography. The enzyme exhibited the enhanced activity at pH 10.0. The enzyme activity totally disappeared in 1.0mM cyanide while it remained 77.8% even in 10 mM azide. These findings indicated that the ensyme was Cu, Zn-SOD type. Molecular mass of the enzyme was estimated to be 34 kDa by gel filtration and 17 kDa on reducing SDS-polyacrylamide gel electrophoresis which indicated a dimer protein.

• Korean Journal of Microbiology
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• v.43 no.1
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• pp.19-22
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• 2007

Acetohydroxyacid synthase (E.C.2.2.1.6., AHAS) is the enzyme that catalyses the first step in the synthesis of the branched-chain amino acids valine, leucine and isoleucine. The AHAS gene (TIGR access code HI2585) from Heamophilus influenzae was cloned into the bacterial expression vector pET-28a and expressed in the Escherichia coli strain BL21(DE3). The expressed enzyme was purified by $Ni^{2+}-charged$ HiTrap chelating HP column. The purified enzyme appears as a single band on SDS-PAGE with a molecular mass of about 63.9 kDa. The enzyme exhibits absolute dependence on the three cofactors FAD, $MgCl_{2}$ and thiamine diphosphate for activity. Specific activity of purified enzyme has 3.22 unit/mg and optimum activity in the pH 7.5 at $37^{\circ}C$. This enzyme activity has an effect on the buffer. When comparing the enzyme activity against the organic solvent, it followed in type and the difference it is but even from the aqueous solution where the organic solvent is included with the fact that the enzyme activity is maintained.