• Journal of Microbiology and Biotechnology
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• v.12 no.3
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• pp.522-525
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• 2002

A very rapid and efficient separation technique for cellular rhizobial cyclosophoraoses was developed based on fractional precipitation and partition chromatography. Cyclosophoraoses are known to function in the osmotic regulation and root nodule formation of legumes during the nitrogen fixation process. Cyclosophoraoses are produced as unbranched cyclic (1longrightarrow12)-${\beta}$-D-glucans in Agrobacterium or Rhizobium species. Recent research has shown that cyclosophoraoses can form inclusion complexation with various unstable or insoluble guest chemicals, thereby implying great potential for industrial application. Typical separation of pure cellular cyclosophoraoses has been so far carried out by several time-consuming steps, including size exclusion, anion exchange, and desalting liquid chromatographies, with a relatively poor recovery. However, the proposed method demonstrated that the successive application of fractional ethanol precipitation and one step of silica gel-based flash column chromatography was enough to simultaneously purify neutral or anionic forms of cyclosophoraoses. This novel technique is very rapid and provides a high recovery.

• Journal of Microbiology and Biotechnology
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• v.20 no.10
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• pp.1415-1423
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• 2010

An extracellular xylanase was purified to homogeneity by sequential chromatography of Fomitopsis pinicola culture supernatants on a DEAE-Sepharose column, a gel filtration column, and then on a MonoQ column with fast protein liquid chromatography. The relative molecular mass of the F. pinicola xylanase was determined to be 58 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis and by size-exclusion chromatography, indicating that the enzyme is a monomer. The hydrolytic activity of the xylanase had a pH optimum of 4.5 and a temperature optimum of $70^{\circ}C$. The enzyme showed a $t_{1/2}$ value of 33 h at $70^{\circ}C$ and catalytic efficiency ($k_{cat}=77.4\;s^{-1}$, $k_{cat}/K_m$=22.7 mg/ml/s) for oatspelt xylan. Its internal amino acid sequences showed a significant homology with hydrolases from glycoside hydrolase (GH) family 10, indicating that the F. pinicola xylanase is a member of GH family 10.

• Journal of the Korean Society of Food Science and Nutrition
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• v.29 no.5
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• pp.908-916
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• 2000

This study was undertaken to purify the anticoagulant polysaccharide from Pachymeniopsis elliptica. The anticoagulant compound (PE-2IVc-2-1) was isolated from the crude polysaccharide (PE-2) by the sequential steps such as ultrafiltration (PE-2IV), anion-exchange chromatography of DEAE-Toyopearl 650C(PE-2IVc), size-exclusion chromatography of Sepharose CL-6B (PE-2IVc), and HPLC Shodex OHpak column chromatography (PE-2IVc-2-1). The apparent molecular weight of purified polysaccharide (PE-2IVc-2-1) was approximately 710 kDa. It was mainly composed of galactose (48.6%) and 3,6-anhydrogalactose (44.0%) with 27.1% of sulfate residue, in addition a trace amount of xylose (3.6%), glucose (1.1%), mannose (1.1%), arabinose (0.8%) and fucose (0.8%). The IR spectrum of the polysaccharide showed the characteristic bands of S=O(1116.74 and 1143.17 $cm^{-1}$) and C-O-S (828.19 $cm^{-1}$) stretching, respectively. The anticoagulant effect of PE-2 in ex vivo was proportional to the concentration of intravenously injected dose, up to 100 mg/kg.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.46 no.4
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• pp.351-358
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• 2013

A protease inhibitor from fish eggs was fractionated using chromatographic methods. The fractionation efficiency was evaluated in terms of specific inhibitory activity (SIA, U/mg), purity (fold), total inhibitory activity (TIA, U), and recovery (%). The protease inhibitor (PI) from egg extracts of skipjack tuna (ST Katsuwonus pelamis), yellowfin tuna (YT Thunnus albacares) and Alaska pollock (AP Theragra chalcogramma) was fractionated using Sephadex G-50 gel filtration and DEAE-Sepharose CL-6B anion exchange chromatography based on protein size exclusion and net charge, respectively. Fractions exhibiting strong inhibitory activity were contained in the 30-50 kDa fraction on gel filtration and in the range of 0.4-0.7 M NaCl gradient fraction on anion exchange chromatography. The respective TIA and percent recovery of the fraction obtained with gel filtration toward trypsin and $N{\alpha}$-benzoyl-L-arginine-p-nitroanilide (BAPNA) were 2,758.7 U and 29.6% for ST, 1,005.5 U and 25.6% for YT, and 1,267.5 U and 26.0% for AP. Gel filtration chromatography was more effective at fractionating PI than using ion exchange chromatography. These results suggest that fish eggs act as serine protease inhibitors and might be useful for protease inhibition in foodstuffs.

• Journal of Korea Technical Association of The Pulp and Paper Industry
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• v.43 no.5
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• pp.60-67
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• 2011

Hemicelluloses pre-extracted from Korean mixed wood chip were investigated as a wet-end additive. Hemicelluloses dissolved in hot water and alkali solution were isolated by ethyl alcohol precipitation from pre-extractives. They showed molecular weight of 9,000 ~ 27,000 g/mol as revealed by size exclusion chromatography. The reduction of molecular weight through hot water extraction was caused by autohydrolysis. Chemical composition of the hemicelluloses were analyzed with high-performance liquid chromatography and UV-Vis spectroscopy. As the surface charge of isolated hemicelluloses were negative, the adsorption of hemicelluloses onto softwood unbleached kraft pulp fiber was promoted by poly-DADMAC. The physical properties of handsheets increased as the molecular weight of hemicellulose increased. On the other hands, the optical property decreased with hemicellulose adsorption.

• Food Science and Biotechnology
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• v.18 no.4
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• pp.872-877
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• 2009

By-products of marine organisms including salmon, skate, flatfish, and yellow goosefish were investigated to search for new source of chondroitin sulfate (CS). Agarose gel electrophoresis with chondroitinase depolymerization showed that purified chondroitin sulfate did not contain any other glycosaminoglycans. 1H-nuclear magnetic resonance (NMR) spectra were acquired to confirm the structure and purity. The average molecular weight ranging from 22 to 64 kDa was determined by high performance size exclusion chromatography. Disaccharide compositions and purities were determined by strong anion exchange-high performance liquid chromatography (SAX-HPLC) after chondroitinase ABC depolymerization. SAX-HPLC data exhibited that the purity was from $81.7{\pm}1.3$ to $114.2{\pm}2.5%$ and the yield was from 1.3 to 12.5%. All analytical results indicate that salmon cartilage, skate cartilage, and yellow goosefish bone could be promising sources of CS to substitute shark cartilage CS in commercial neutraceuticals.

• Journal of Microbiology and Biotechnology
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• v.19 no.12
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• pp.1620-1627
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• 2009

The design and expression of an antihypertensive peptide multimer (AHPM), a common precursor of 11 kinds of antihypertensive peptides (AHPs) tandemly linked up according to the restriction sites of gastrointestinal proteases, were explored. The DNA fragment encoding the AHPM was chemically synthesized and cloned into expression vector pGEX-3X. After an optimum induction with IPTG, the recombinant AHPM fused with glutathione S-transferase (GST-AHPM) was expressed mostly as inclusion body in Escherichia coli BL21 and reached the maximal production, 35% of total intracellular protein. The inclusion body was washed, dissolved, and purified by cation-exchange chromatography under denaturing conditions, followed by refolding together with size-exclusion chromatography and gradual dialysis. The resulting yield of the soluble GSTAHPM (34 kDa) with a purity of 95% reached 399 mg/l culture. The release of high active fragments from the AHPM was confirmed by the simulated gastrointestinal digestion. The results suggest that the design strategy and production method of the AHPM will be useful to obtain a large quantity of recombinant AHPs at a low cost.

• Journal of Microbiology and Biotechnology
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• v.15 no.3
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• pp.560-567
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• 2005

Three groups of exopolysaccharides (EPSs) (designated as Fr-I, Fr-II, and Fr-III) were isolated from the culture filtrates of Paecilomyces sinclairii by gel filtration chromatography on Sepharose CL-4B. Their molecular characteristics were examined by multi-angle laser light scattering (MALLS) connected online to a size exclusion chromatography (SEC) and refractive index (RI) detector system. The weight-average molar mass of Fr-I, Fr-II, and Fr-III of EPSs were determined to be $1.540{\times}10^6,\;6.302{\times}10^4$, and $9.389{\times}10^4\;g/mol$, respectively. All three EPSs showed a fairly low polydispersity indice, ranging from 1.008 to 1.059 (nearly mono disperse behavior), and showed different carbohydrates and amino acids compositions; all fractions of EPSs consisted of mainly cystine, valine, and arginine in the protein moiety, and mainly ribose, galactose, and glucose in the carbohydrate moiety. The determination of gyration radii of the EPSs in SEC/MALLS analysis revealed the molecular shape of the Fr-I to be a rod-like structure, whereas the Fr-II and Fr-III had a random-coil structure in an aqueous solution.

• Macromolecular Research
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• v.12 no.4
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• pp.325-335
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• 2004

Molecular weight distributions of poly(vinyl neo-decanoate) produced by the bulk polymerization of the monomer to low conversions were investigated to obtain values of the rate constants for the chain transfer to monomer ( $C_{M}$). The value of $C_{M}$ of 7.5($\pm$0.6)${\times}$10$^{-4}$ was obtained from a logarithmic plot of the number distribution at 5,25, and 5$0^{\circ}C$, which suggests that the activation energy for chain transfer is on the order of 20-25 kJ ㏖$^{-1}$ . These plots were linear between the number and weight-average degrees of polymerization, but not over the whole molecular weight range for which a significant signal was observed in the gel permeation chromatography (GPC) trace. Modeling suggests that the deviations observed at high molecular weights can be explained by branching of the chains through chain transfer to the polymer, with a branching density as low as 10$^{-5}$ , without affecting the slope at low values of the number of monomer unit, N. This deviation from the expected distribution of linear chains was used to estimate the branching densities at low conversion.ion.

• Journal of Applied Biological Chemistry
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• v.43 no.4
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• pp.235-239
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• 2000

A polysaccharide with antithrombin activity in Manda$^{(R)}& (PAM) was purified via procedures comprising three major steps, i.e. fractional precipitation with ethanol, anion exchange chromatography, and gel permeation chromatography. PAM showed a symmetrical peak on size exclusion HPLC, as assessed by refractive index, and behaved as a single band on cellulose acetate electrophoresis. The average molecular mass was estimated to be 222 kDa by gel filtration. PAM was found to be a sulfated heteropolysaccharide that contains sulfate group (20.5%, w/w) and uronic acid moiety (7.1 %, w/w) in addition to neutral sugar consisting of fucose, xylose, mannose, galactose, and glucose in a molar ratio of 1.00 : 0.35 : 0.28: 0.22 : 0.15. This polysaccharide appeared to inhibit blood coagulation via the intrinsic pathway in a dose-dependent pattern. The clotting of fibrinogen by thrombin was also significantly mitigated by the presence of PAM.