• KSBB Journal
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• v.15 no.1
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• pp.42-48
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• 2000

Urokinase (UK), a thrombolytic enzyme used to clear catheters obstructed by blood clots, can be also used industrially in the recombinant protein purification system to cleave a fusion protein linked with a certain fragment of GST. We have immobilized UK by covalent attachment to activated Sepharose 6B-Cl gels and evaluated its performance to cleave a fusion protein of hGH and GST. The Sepharose gels were activated by etherification with glycidol (2,3-epoxypropanol) and further oxidized with periodate resulting in glyceryl-Sepharose gels. After the activation treatment, surface density of the aldehyde groups was 7-30 $\mu$mol-aldehde/mL-gel. Immobilization yield was higher than 99% at high pH (10.5), and the immobilized UK maintained ca. 80% specific activity of the soluble UK. In a column reaction the cleavage yield heavily depended on the feed rate, and it was nearly 86% of that from soluble UK. And the immobilized UK was successfully regenerated by unfolding and refolding with 6M GuHCl. After cleavaging reaction, the monomeric hGH was purified by using expanded bed adsorption chromatography.

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 1986.12a
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• pp.526.2-526
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• 1986

P. mirabilis 로부터 정제한 cysteinylglycine 분해효소의 성질 및 세포내 국재성을 검토하였다. 본 효소의 일반적 성질은 pH가 7.3 온도 37$^{\circ}C$에서 최대 활성을 냐타내었으며 pH 8.0에서 안정하였고, 열안정성은 $50^{\circ}C$, 30분처리에 30%의 활성 손실을 보였다. 또한 $Mn^{+2}$이온과 $Mg^{+2}$ 이온에 의해 활성이 촉진되었으며 본 효소를 반응전 30분 Preincubation 하므로서 최대 활성을 보였다. 본 효소는 glutathione 일단계 분해효소인 ${\gamma}$-glutamyl transpeptidase와 마찬가지로 세포내의 periplasmic space에 존재하였다.

• Microbiology and Biotechnology Letters
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• v.44 no.3
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• pp.333-340
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• 2016

An alkaline protease was purified and characterized from an alkalophilic microorganism, Bacillus sp. DK1122, isolated from soil in central Korea. The optimum temperature and pH for the growth of the producer strain were 40℃ and pH 9.0, respectively. The protease was produced aerobically at 40℃ after 24 h incubation in modified Horikoshi I medium (pH 9.0) containing 0.5% (w/v) glucose, 0.8% (w/v) yeast extract, 0.5% (w/v) polypeptone, 0.1% (w/v) K₂HPO₄, 0.02% (w/v) MgSO₄·7H₂O, 1% (w/v) Na₂CO₃, and 3% (w/v) NaCl. The alkaline protease was purified by 70% ammonium sulfate precipitation of the culture supernatant of Bacillus sp. DK1122, followed by CM-Sepharose chromatography. The molecular weight of the enzyme was estimated to be 27 kDa on the basis of SDS-PAGE. The optimum temperature and pH for the protease activity were 60℃ and pH 9.0, respectively. Addition of CaCl₂ increased the thermal stability of the purified protease, where 90% of protease activity was retained at 60℃ for up to 3 h. Consequently, it is expected that the alkaline protease from this study, exhibiting stability at pH 7–9 and 60℃, may be promising for application in the food and detergent industries.

• The Korean Journal of Mycology
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• v.22 no.4
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• pp.298-308
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• 1994

The properties of polygalacturonase by Ganoderma lucidum in liquid culture were investigated. The enzyme was composed of an endo- and an exo-polygalacturonase. The endo- and exo-polygalacturonase were purified approximately 56 and 9.2-fold, respectively, through ammonium sulfate fractionation, gel filtration on Biogel P-100, anion exchange chromatography on DEAE-cellulose, gel chromatography on Sephadex G-150 and re-gel chromatography on Sephadex G-150. The endo- and exo-polygalacturonase had higher affinity for apple pectin than for citrus pectin or pectic acid. The Km values of the endo- and exo-polygalacturonase for apple pectin, determined on the Lineweaver-Burk plot, were 1.44 and 10.6 mg $ml^{-1}$ for apple pectin, respectively. Purified endo-polygalacturonase was found to be homogeneous electrophoretically and had a molecular weight of 54,000 estimated on SDS polyacrylamide gel. The optimal pH for the activity of the enzymes was 4.0. The endo- and exo-polygalacturonase were stable in the pH range of 4.0 to 6.0 and 3.5 to 5.5, respectively. The optimal temperatures of the endo- and exo-polygalacturonase were 40 and $60^{\circ}C$, respectively. The exo-polygalacturonase was more resistant to heat than the endo-polygalacturonase, requiring heating for 40 min at $80^{\circ}C$ for complete inactivation. The activity of the endo-polygalacturonase was increased by $Ca^{++}$ and $Mn^{++}\;ions$, while that of the exo-polygalacturonase was increased by $Ca^{++}\;ion$ only, and was not affected by $Mn^{++}\;ion$.

• Microbiology and Biotechnology Letters
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• v.20 no.5
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• pp.551-558
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• 1992

The extracellular endoinulase from Streptomyces sp. 556 was purified and characterized, The culture broth was fractionated by ammonium sulfate saturation followed by DEAE-cellulose column chromatography and 5ephadex G-200 gel filtration, The ultimately purified fraction revealed a single band in 7.5% polyacrylamide gel electropherogram. The purified enzyme showed the maximal activity at pH 5.5-6.0 and $50^{\circ}C$, but lost 93% of inulase activity after 30 min incubation at $55^{\circ}C$ . The essen.tial amino acid residue for catalytic activity appeared to be tryptophan. This endo inulase was activated by $Mn^{2+}$, whereas inactivated by $Ag^{+}$, $Hg^{+}$, $Cu^{2+}$, $Zn^{2+}$, $Fe^{3+}$ and $Mo^{6+}$ EDTA and 8-hydroxyquinoline inhibited the enzyme so that the enzyme was considered to be a metalloenzyme. The Km value for inulin was 0.287 mM, and no invertase or $\alpha$-glucosidase activity was found in the enzyme.

• Journal of the Korean Society of Food Science and Nutrition
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• v.33 no.3
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• pp.560-565
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• 2004

Bacteriocin producing lactic acid bacteria (LAB) were isolated from Kimchi by using spot-on-the-lawn method. Listeria monocytogenes, Staphylococcus aureus, and Lactobacillus plantarum were used as indicators. One isolate (P3-l) produced a bacteriocin efficiently inhibiting the growth of Listeria monocytogenes. 16S rDNA sequence and sugar utilization test identified that P3-1 was a Lactobacillus sakei strain. Accordingly, the isolate was named as Lactobacillus sakei P3-1. L. sakei P3-1 produced a bacteriocin which efficiently inhibited the growth of Listeria monocytogenes but did not inhibit other Gram positive and negative organisms tested. The bacteriocin was stable against heat, organic solvent, and pH variation and it retained 50% of activity after 10 min heat treatment at 10$0^{\circ}C$. The molecular weight of Sakacin P3-1 was estimated to be 4 kDa by SDS-PAGE.

• Applied Biological Chemistry
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• v.35 no.5
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• pp.375-381
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• 1992

An exoinulase-producing bacterium was isolated from soil, and identified as Streptomyces sp. The maximum inulase production was achieved when inulin as carbon source and soybean meal as organic nitrogen source were included in the culture. The exoinulase was considered to be a constitutive enzyme produced not only by inulin but also by soluble starch or glucose. The purified enzyme on DEAE-cellulose and Sephadex G-200 column showed the maximal activity at $pH\;5.5{\sim}6.0$ and $50^{\circ}C$, but lost 65% inulase activity at $50^{\circ}C$ after 1 hour incubation. This exoinulase was activated by $Mn^{+2}$, wherease more that 80% inactivation was observed with $Ag^+$, $Hg^{+2}$ and $Fe^{+3}$. The enzyme was possibly a metalloenzyme in that EDTA and 8-hydroxyquinoline inhibited the enzyme. Km values for inulin (16.51 mM) and sucrose (14.62 mM) were in very close range suggesting mostly equal affinity toward the subatrates. However, the maximum velocity for inulin was 10 times greater than for sucrose.

• Microbiology and Biotechnology Letters
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• v.2 no.3
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• pp.133-140
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• 1974

1) Two xylanase (designed as A and B) of Myriococcus albomyces were purified from an extract of wheat koji culture. Puriscation steps included first ammonium sulfate fractionation followed successively by SE-Sephadex column chromatgraphy. DEAE-Sephadex column chromatography and gel filtration on Sephadex G-100 repectively. 2) The optimum pH and pH stability for crude xylanse were found to be pH 5.0 and pH 4.0-7.0 respectively. 3) The optimium temperature was found to he 5$0^{\circ}C$ and for the thermal statbility of xylanase, the enryme incubated at $65^{\circ}C$ for 60min did not affect their stability. 4) The purised xylanase A and B were considered as liquefying xylanase and saccharogenic xylauase repectively. 5) The Bylanase A was most active at pH 4.0 and range of pH 3.0-8.0 at 3$0^{\circ}C$ for six hrs. The B was most active at pH 5.0 showing stability range of pH 4.0 to 8.5 at 3$0^{\circ}C$ for 6 hrs. incubation respectively. The Optimum temperature of xylanase A and B were found to be 7$0^{\circ}C$ and $65^{\circ}C$ for 60min repectively.

• Korean Journal of Food Science and Technology
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• v.35 no.5
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• pp.791-796
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• 2003

Polyphenol oxidase isoforms were purified from the lotus roots using 50% acetone precipitation, conventional chromatographies of Q-Sepharose and hydrophobic interaction, and high performance liquid chromatographies of Mono-Q and Superdex 75 gel-filtration. Molecular mass of a purified PPO isoform (LPIII-2) was determined to be 56 kDa using gel-filtration chromatography. The active form of LPIII-2 appeared to bea heterodimer, as purified LPIII-2 on SDS-PAGE gel showed two bands that were determined to be 28 kDa and 26 kDa. To further characterize PPO, partially purified PPO isoforms (LP-II, LP-III) were obtained from Q-Sepharose anion-exchange chromatography. In substrate specificity, the partially purified PPO isoform LP-II showed a high affinity to catechol, while LP-III showed a high affinity to pyrogallol. The optimum pH of LP-II and LP-III was pH 7.0. Interestingly, the partially purified PPO isoforms showed high activities at low temperatures $(0{\sim}5^{\circ}C)$, and as temperatures rose, the activities decreased. Both PPO isoforms were stable at $40^{\circ}C$ and were inactivated by incubation at $60^{\circ}C$ for 40 min.

• Journal of the Korean Society of Food Science and Nutrition
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• v.16 no.2
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• pp.110-117
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• 1987

Polyphenol oxidase in Prunus salicina(Red) was extracted, some properties and its partially purification were investigated as follows; Polyphenol oxidase was purified about 15 folds after ammonium sulfate saturation and about 64 folds after Sephadex G-100 column chromatography. Polyphenol oxidase showed optimum pH for activity at 6.5 and optimum temperature at at $35^{\circ}C$ and high affinity to catechol in o-diphenol compounds. Thermal stability were about 85% and 75% of initial polyphenol oxidase activity remained after heating at $50^{\circ}C$ for 5 minutes and 30 minutes respectively. The Michaelis constant of the enzyme was 2.58mM. L-cysteine, glutathione, ascorbic acid and potassium cyanide appeared to be most effective inhibitors. EDTA showed a slight inhibition.