• Preventive Nutrition and Food Science
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• v.9 no.1
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• pp.21-28
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• 2004

We purified and characterized a crude polysaccharide from Spirodela polyrhiza with anti-complement activities. The crude polysaccharide fraction (SP-0) which had potential anti-complement activity was extracted in hot water for 4 hrs at 10$0^{\circ}C$. The ethanol-precipitate, the crude polysaccharide traction (SP-1), showed a potent anti-complement activity. Further purification of the crude polysaccharide (SP-1) was carried out by cetavlon, ion exchange chromatography and gel column chromatography. Among cetavlon fractions, SP-4 showed the most potent anti-complement activity. When 100 $\mu\textrm{g}$/mL of SP-4 was incubated with an equal volume of normal human serum (NHS), the TCH$_{50}$ was reduced by about 78%. When the SP-4 fraction was further purified by DEAE-Sepharose (Cl$^{[-10]}$ ), the SP-4IIa, SP-4IIb and SP-4IIc, absorbed fractions, were almost the same as the anti-complement activities of SP-4. SP-4IIc, having the greatest potential activation and the highest yield by ion exchange chromatography, was further purified by gel column chromatography on a Sepharose CL-6B column. Four polysaccharide fractions of SP-4IIc-1, SP-4IIc-2, SP-4IIc-3 and SP-4IIc-4 were obtained, consisting mainly of arabinose, rhamnose, galactose and glucose, with approximate molecular weights of about 305,000, 132,000, 64,000 and 12,000, respectively. Among these subfractions, SP-4IIc-1 had the most potent anti-complement activity. When the SP-4IIc-1 aggregate was applied to a gel column chromatography in 10 mM and 50 mM NaCl solution, the position of the peak fractions shifted to a low molecular weight region, and the molecular weight of SP-4IIc-1 decreased with increased NaCl concentration in the gel column chromatography. It was found that the self-aggregation formed spontaneously in void volume by gel column chromatography using Sepharose CL-6B in water and the self-aggregation significantly affected the anti-complement function.

• Applied Biological Chemistry
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• v.49 no.3
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• pp.180-185
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• 2006

${\alpha}$-Galactosidase was partially purified from the culture filtrate of Debaryomyces sp. by Mannobiose-Sepharose affinity column chromatography. The galactosidase exhibited maximum activity at pH 4.0 and $60^{\circ}C$, and was stable in the pH and temperature ranges of 3 to 4.5 and 30 to $50^{\circ}C$, respectively. The enzyme was inhibited by $Hg^{2+}\;and\;Ag^{2+}$. The enzyme activity was not affected considerably by treatment with other metal compounds. The enzyme hydrolyzed melibiose to galactose and glucose, raffinose to galactose and sucrose, and $Gal^3Man_3$ ($6^3-{\alpha}$-galactosyl-1,4-mannotriose) to galactose and mannotriose. On the contrary, it could not hydrolyze $Gal^3Man_4$ ($6^3-{\alpha}$-galactosyl-1,4-mannotetraose).

• Microbiology and Biotechnology Letters
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• v.26 no.5
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• pp.413-419
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• 1998

Aryl acylamidase [EC 3.5.1.13] present in an acetaminophen-assimilating Pseudomonas sp. has been purified to a homogeneity using series of ammonium sulfate fractionation, DEAE-Sephacel anion exchange, Phenyl-Sepharose CL-4B hydrophobic, and Sephadex G-100 gel-permeation chromatography. The molecular weight, which was estimated by gel-permeation filtration and sodium dodecyl sulfate polyacylamide gel electrophoresis, was about 57 kDa and 56 kDa, respectively, indicating that this enzyme is a monomeric protein. The optimum pH was 10.5 and the optimum temperature was 40$^{\circ}C$. After incubation of the enzyme at 50$^{\circ}C$ for 30 min, residual activity of the enzyme was 34% compared to its original activity. The Km values for acetaminophen and 4'-nitroactanilide were 0.10 mM and 0.11 mM, respectively.

• KSBB Journal
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• v.14 no.2
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• pp.192-197
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• 1999

marine bacterium Pseudomonas sp. CHCS-2 produced the biosurfactant in the culture broth which contained 2%(w/v) arabian light crude oil and the productivity of biosurfactant was increased with the addition of glucose. The crude oil in the culture broth was degraded by this strain and carbon chain of $_nC_{12}~_nC_{22}$ was completely degradaded during the incubation for 196 h. The crude biosurfactant was purified by Amberlite XAD-7, Sepharose CL-4B and DEAE-Sepharose CL-6B column chromatography. Therefore, 0.21g/L of the purified biosurfactnat was obtained. The purified biosurfactant was a type of lipoprotein and the molecular weight was estimated as 67kDa by SDS-PAGE. The lipid composition was identified as octadecanoic acid by gas chromatography/mass spectrometry. And then, the N-terminal amino acid sequence of the protein was determined as Ser-Val-lle-Asn-Thr-lle-X-Met-lle-Gly-Gln-Gln- and the sequence did not show homology to any other known lipoprotein. Therefore, the purified lopoprotein was predicted novel biosurfactant.

• Journal of Life Science
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• v.12 no.4
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• pp.483-489
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• 2002

We investigated the biochemical properties of insect defensin expressed and secreted from Saccharomyces corevisiae. The defensin showed extremely high resistance to boiling for up to 30 min and to pH values tested from 2.0 to 12.0. The treatment of defensin with various proteases abolished antibacterial activity. However, amylases, cellulase, lipase and catalase had no effect on the activity. The defensin was purified to homogeneity through ammonium sulfate concentration of culture supernatant, SP-Sepharose column chromatography and RP-HPLC. Tricin-SDS-PAGE analysis revealed that the molecular weight of the defensin was about 4.0 kDa. The antibacterial activity of the purified defensin was verified by renaturation of stained gel and gel pouring assay using Micrococcus luteus as a test organism.

• Korean Journal of Microbiology
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• v.43 no.2
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• pp.83-90
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• 2007

Sphingomonas sp. KS 301, which was isolated from oil contaminated soil, was shown to have five different SODs (SODI, II, III, IV, V) which can be separated by DEAE-Sepharose chromatography, and SOD III was finally purified in this study by ammonium sulfate precipitation, DEAE-Sepharose chromatography, Superose 12 gel filtration and Uno-Q1 ion exchange chromatography. The molecular weight of SOD III was 23 kDa as determined by SDS-PAGE and the apparent molecular weight of the native enzyme was estimated to be approximately 71 kDa by Superose-12 gel filtration chromatography. These data suggest that the purified SOD consists of at least two subunits. The specific activity of the SOD III was higher than Mn type or Fe type SOD of Escherichia coli by 5 fold. To determine the type of SOD III, inhibitory effects of $NaN_{3},\;H_{2}O_{2},\;KCN$ were examined. 10 mM $NaN_{3}$ was able to inhibit 56% of the SOD III activity, which indicates that this SOD is Mn type. The optimum pH of the SOD III was 7.0 and the optimum temperature was $20^{\circ}C$. N-terminal amino acid sequence of purified SOD III was most similar to those of Psudomonase ovalis and Vibrio cholerae among bacteria.

• Journal of Life Science
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• v.17 no.5 s.85
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• pp.625-633
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• 2007

We isolated a new extracellular alginate lyase-producing microorganism, which displayed alginate-depolymerizing activity in plate assays, from coastal soils in Wando, Jeollanam-do, Korea. This alginate-depolymerizing bacterium belonged to the genus Streptomyces and it was named Streptomyces sp. MET 0515. An extracellular alginate lyase(ALY1) secreted by Streptomyces sp. MET 0515, was purified to homogeneity by a combination of acetone precipitation, anion-exchange chromatography (Q-Sepharose and DEAE-Sepharose) and Sephacryl S-200 HR gel filtration chromatography. Its molecular mass was 26 kDa as determined by SDS-PACE analysis. The enzyme had an optimal temperature of $70^{\circ}C$ for its activity, and was most active at pH 7.5. The thermal and pH stability were $0-50^{\circ}C$, and pH 6.0-9.0, respectively. The enzyme activity was stimulated by 1mM $Mn^{2+}$, and inhibited by 1mM $Fe^{3+}$, 1mM EDTA and 1mM $Zn^{2+}$. Preliminary analysis of substrate specificity showed that this alginate lyase had activity on both poly-alpha 1,4-L-guluronate and poly-beta 1,4-D-mannuronate in the alginate molecule.

• Applied Biological Chemistry
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• v.37 no.5
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• pp.334-338
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• 1994

Streptococcal nuclease was completely purified by stepwise CM-Sepharose column chromatography from crude extracts isolated from Streptococcus sp. The active enzyme fraction was eluted with the buffer containing 0.2 M NaCl. The purified enzyme showed a homogeneity on SDS PAGE and had a molecular weight of 35,000 daltons. The optimum pH and temperature for the enzyme were 9.0 and $50^{\circ}C$, respectively.

• Microbiology and Biotechnology Letters
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• v.37 no.2
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• pp.147-152
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• 2009

A xylanase was purified from the culture supernatant of Bacillus sp. A-6 by using ultrafiltration and ion exchange chromatography on the column of SP-Sepharose using 5 mM acetate buffer, pH 5.0. The xylanase was eluted from the column at the concentration less than 0.05 M NaCl. The eluted xylanase was shown to be a single protein band in SDS-PAGE. Zymogram analysis indicated that the protein band in SDS-PAGE had the enzyme activity to hydrolyze oat spelt xylan. The molecular weights of the xylanase were 15,000 based on SDS-PAGE and 14,100 based on gel filtration chromatography. Thin layer chromatography showed that the xylanase hydrolyzed oat spelt xylan into xylobiose and high-molecular-weight xylooligosaccharides. The relative activities of the heated xylanase decreased to 80% at $40^{\circ}C$ after 7 hr and less than 40% at $60^{\circ}C$ after 1 hr.

• Applied Biological Chemistry
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• v.35 no.3
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• pp.173-178
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• 1992

A bacterium having CMCase activity was isolated form soil and identifed as a Pseudomonas sp YD-15. The optimum conditions for the production of CMCase were avicel 1.2%, yeast extract 0.5%, $KNO_3$ 0.06%, $K_2HPO_4$ 0.2%, $MgSO_4$ $7H_2O$ 0.15%, pH 8.0, $30^{\circ}C$ and 60 hours cultivation. The CMCase was purified 15.2 folds with 14ft yield through ammonium sulfate precipitation, DEAE-sepharose column chromatography and sephadex G-100 gel filtration chromatography. The optimum pH and temperature for the enzyme activity were 6.0 and $50^{\circ}C$, respectively. The enzyme was stable between pH 5.0 and 8.0, below $50^{\circ}C$. The molecular weight was calculated about 100,000 by SDS-polyacrylamide gel electrophoresis. $K_m$ value for CMC used as a substrate was 40 mg/ml.