• Journal of the Korean Wood Science and Technology
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• v.35 no.5
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• pp.100-108
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• 2007

$\beta$-Glucosidase from Laetiporus sulphureus among the enzymes related to lignocellulosic biomass degradation to sugars for using alternative bioethanol production was characterized. The highest activity of $\beta$-glucosidase was obtained on cellobiose at shaking culture. For the characterization and purification of $\beta$-glucosidase culture solution was concentrated and then purified by FPLC using ion exchange and size exclusion column. According to the results of SDS-PAGE, native PAGE and microfluidic system of purified enzyme, protein band was observed at about 132 kDa. Optimal pH and temperature of purified $\beta$-glucosi-dase were 5.0 and $60^{\circ}C$, respectively. In the kinetic properties of $\beta$-glucosidase on various substrates such as sophorose, gentiobiose and cellobiose, $K_m$ was 0.81, 1.07 and 1.70 mM, respectively.

• Journal of Life Science
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• v.6 no.4
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• pp.250-263
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• 1996

In this study Carotenoprotein from Styela clava were extracted, and purified by ammonium sulfate fraction, Sephadex G- 200 gel chromatography and DEAE-cellulose ion exchange chromarography. The purified carotenoprotein from styela clava had absorption maxima of 487nm, 463nm and 280nm, and the carotenoid liberated from carotenoprotein had 478nm and 452nm with inflexion. One miligram of caroteno-protein contained 0.35 $\mu$g of carotenoid. The carotenoprotein had an approximate molecular weight of 398 kDa (gel filtration). SDS-PAGE showed only a single polypeptide chain with a molecular weight of 62.4 kDa. The amino acid composition of the carotenoprotein were mainly glutamic acid(11.48%), valine(10.75%), leucine (10.45%), aspartic acid(9.94%), while cysteine and tryptophan were not found. The carotenoprotein contained lipid as structure units. In the carotenoprotein, the major fatty acids were oleic acid, plamitoleic acid and palmiunsaturated fatty acids 33.6%, saturated fatty acids 18.6%. In addition, the levels of higher unsaturated fatty acids were high as much as 30.8% of the total fatty acids. Carotenoid was extracted from the carotenoprotein by acetone. Thin layer chromatography showed only carotenoid to be present. Its chemical reactivities and spectroscopic properties were studied and elucidated as astaxanthin.

• Microbiology and Biotechnology Letters
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• v.23 no.4
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• pp.454-460
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• 1995

Acetylxylan esterase II was produced by Escherichia coli HB101 harboring the recombinant plasmid pKMG7 which contained the estII gene of Bacillus stearothermophilus. Optimal medium for the production of the acetylxylan esterase by E. coli HB101/pKMG7 was determined to contain 0.5% galactose, 1% yeast extract and 1% NaCl. The enzyme produced was purified to homogeneity using a combination of 20-50% ammonium sulfate precipitation, DEAE-Sepharose CL-6B chromatography and Sephacryl S-200 gel filtration. The temperature and pH optimum of the esterase were 45$\circ$C and pH 6, respectively. The essential amino acids for the esterase activity were found to be methionine, serine, and cysteine. Molecular weight of the esterase was determined to be 28 kDa by SDS-polyacrylamide gel electrophoresis, and 120 kDa by gel filtration. This suggests that the functional enzyme is a homomeric tetramer. The esterase had an isoelectric point of pH 3.4. The N-terminal amino acid sequence of the enzyme was Ala-Leu-Phe-Glu-Ser-Arg-Phe-Phe-Ser-Glu-Val-Leu-Gly-Leu.

• Microbiology and Biotechnology Letters
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• v.19 no.6
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• pp.592-597
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• 1991

An extracellular xylanase of Bacillus stearothemophilus was purified to a single protein through a sequency of operations including ammonium sulfate fractionation, DEAE Sepharose CL-6B ion exchange chromatography, Sephadex G-100 gel filtration and heat treatment. The purified enzyme had a moleular weight of 170, 000. the pH and temperature optima for the enzyme activity were pH 9.0 and $55^{\circ}C$, respectively. The activity was enhanced by $co^{2+} \; and\; Mn^{2+}$, and inhibited by $Hg^{2+}$. Pattern of hydrolysis demonstrated that the xylanase was an endo-splitting enzyme able to break down larchwood xylan at random giving xylobiose and xylotriose as the main end products.

• Journal of Life Science
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• v.5 no.4
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• pp.170-180
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• 1995

A carotennnoprotein from the skin of Ascidian(Halocynthia roretzi) was extracted by Triton X-100 and purified by ammonium sulfate fraction, SephadexG-200 charomatography and DEAE-cellulose ion exchange chromatography. The carotenoprotein was redwith broad $\lambda$$_{max}$ between 495, 467 and 318nm. The red carotenoprotein had an approximate molecular weight of 326KDa(gel filtration). SDS-PAGE indicated the presence of two polypeptodes of 84.1KDa and 74.4KDa, with different mobility in polyacrylamide gel electrophoresis. In the presence of denaturing agents such as organic solvent aand extreme pH, the red complex readily disociates to liberate the yellow carotenoid($\lambda$$_{max}$ 452nm) and a colourless apoprotein. The amino acid composition of carotenoprotein were mainly threonine(15.2%), aspartic acid(12.2%), glutamic acid(11.9%) and serine(9.6%), while proline was not found. The carotenoprotein consisted of lipids as structure units. Its major fatty acids composion were C$_{18:1}$, C$_{16:1}$, and C$_{16:0}$. The monounsaturated fatty acids(41.5%) contained abundant content compared to other fatty aacids(polyunsaturated fatty acids 37.4%, saturated fatty acids 20.6%).

• Journal of Microbiology and Biotechnology
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• v.20 no.12
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• pp.1681-1688
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• 2010

A screening for cellobiohydrolase (CBH) activity was performed and Fomitopsis pinicola KMJ812 was selected for further characterization as it produced a high level of CBH activity. An extracellular CBH was purified to homogeneity by sequential chromatography of F. pinicola culture supernatants. The molecular mass of the F. pinicola CBH was determined to be 64 kDa by SDS-PAGE and by size-exclusion chromatography, indicating that the enzyme is a monomer. The F. pinicola CBH showed a $t_{1/2}$ value of 42 h at $70^{\circ}C$ and catalytic efficiency of $15.8mM^{-1}s^{-1}(k_{cat}/K_m)$ for p-nitrophenyl-${\beta}$-D-cellobioside, one of the highest levels seen for CBH-producing microorganisms. Its internal amino acid sequences showed a significant homology with hydrolases from glycoside hydrolase family 7. Although CBHs have been purified and characterized from other sources, the F. pinicola CBH is distinguished from other CBHs by its high catalytic efficiency and thermostability.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1997.04a
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• pp.93-93
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• 1997

Analysis of protein is often frustrated by the inability to isolate large amounts of purified protein from a native source. To overcome this problem, fusion protein expression systems such as pGEX system have been widely used. Using pGEX system, the desired protein could be easily obtained in a large amount in E. coli, and then the fusion protein could be used for the study of the function of the given protein. To analyze and purify the GST fusion protein, anti-GST antibody could be used as one of the system of choice. However, the production and characterization of monoclonal anti-GST antibody has not been studied extensively yet. To produce monoclonal anti-GST antibody, GST was purified from E. coli transformed with pGEX-cs, one of the pGEX system and was used as an antigen. The monoclonal antibody was produced by fusion of the immunized spleen cells with SP2-0 myeloma cells. The antibody was characterized by ELISA, western blotting, etc. The monoclonal antibody produced in this study (mAb-GSTA) showed strong and specific immunoreactivity against not only GST but also GST-fusion proteins. Also, mAb-GSTA was successfully used for the immunoaffinity purification of the GST ${\beta}$-Rc.-third intracellular-loop fusion protein. The results of the present study suggest that mAb-GSTA may be used for the identification and purification of GST fusion proteins.

• Bulletin of the Korean Chemical Society
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• v.32 no.12
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• pp.4253-4257
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• 2011

Sodium dodecyl sulfate-capillary gel electrophoresis (SDS-CGE) and capillary isoelectric focusing (CIEF) were employed to characterize and compare ricin E purified from the small grain seeds of Ricinus communis with ricin D isoform. During the purification of ricin E using ion-exchange and size-exclusion chromatography, SDS-CGE was found to be useful for monitoring the efficiencies of purification steps. SDS-CGE showed that the molecular size of ricin E was not significantly different from that of ricin D, which was confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. CIEF was useful for discriminating ricin E from ricin D based on their isoelectric points (pI). The pI values of ricin E and D were 8.6-8.8 and 7.0-7.4, respectively. This study demonstrates the usefulness of SDS-CGE and CIEF for the characterization of ricin toxins.

• KSBB Journal
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• v.14 no.3
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• pp.322-327
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• 1999

The cholesterol oxidase(EC.1.1.3.6) produced from Streptomyces sp. No.4 which isolated from soil was purified and investigated for the enzymatic properties. The enzyme was purified specifically by cholesterol affinity column chromatography with a yield of 28.3%. The purified enzyme showed a single polypeptide on SDS-PAGE and the molecular weight was estimated to be 60,000 daltons. The enzyme activity was strongly inhibited by metal ions such as $HgCl_2$ and $CuSO_4$. Dithiothreitol and mercaptoethanol inhibited the enzyme activity at concentration of 1mM. The Michaelis constant(Km) for cholesterol was found to be 1.38mM by Lineweaver-Burk plot analysis. Amino acid analysis showed that the enzyme protein was composed of 416 amino acid residues including 52moles of glycine and 19moles of tryptophane.

• BMB Reports
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• v.29 no.5
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• pp.455-461
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• 1996

A serine proteinase was purified from Acanthamoeba culbertsoni by 41~80% ammonium sulfate fractionation, ion exchange chromatography, affinity chromatography and gel filtration chromatography. The molecular weight of the purified enzyme was estimated to be 108.0 kDa by gel filtration chromatography and 54.0 kDa by SDS-PAGE. Therefore, the purified enzyme seemed to be a dimer. Isoelectric point was 4.5. The enzyme activity was highly inhibited by the serine proteinase inhibitors diisopropyl fluorophosphate (OFP) and phenylmethyl sulfonylfluoride (PMSF). It had a narrow pH optimum of 6.5~7.5 with a maximum at pH 7.0. These data suggested that the purified enzyme was a neutral serine proteinase. Optimal temperature was $37^{\circ}C$. It was stable for at least 16 h at $4^{\circ}C$ and $37^{\circ}C$, but it was rapidly inactivated at $65^{\circ}C$ The activity of the purified enzyme was not influenced significantly by $Mg^{2+}$, $Mn^{2+}$, $Zn^{2+}$ or $Ca^{2+}$. However, the enzyme activity was highly inhibited by $Hg^{2+}$ The enzyme degraded type I collagen and fibronectin, but not BSA, hemoglobin, lysozyme, immunoglobulin A or immunoglobulin G.