Purification and characterization of intracellular cellulase produced by A. oryzae ITCC-4857.01 are reported. The enzyme was purified by ion-exchange chromatography using DEAE-cellulose followed by Gel filtration. The purification achieved was 41 fold from the crude extract with yield of 27%. The purified enzyme showed single band on poly acrylamide gel. The molecular weight as determined by SDS-PAGE and gel filtration was 38 KDa and 38.6 KDa respectively and contained only one subunit. The enzyme is glycoprotien as nature and contained 0.67% neutral sugar. The apparent Km value of the enzyme against cellulose was 0.83%. The enzyme showed the highest relative ativities on CMC followed by avicel, salicin and filter paper. The optimum pH of activity was 5.5 and very slight activity was observed at or above pH 7.5 as well as bellow pH 3.5. The optimum tempreture of the activity was 45degrees C and the highest activity was exhibited in 35 to 45degrees C. The enzyme lost their activities almost completely (95~100%) at 80 degrees C or above and as well as bellow 25degrees C.
Acrylamide ; Aspergillus ; Aspergillus oryzae ; Benzyl Alcohols ; Cellulase ; Cellulose ; Chromatography, DEAE-Cellulose ; Chromatography, Gel ; Chromatography, Ion Exchange ; DEAE-Cellulose ; Electrophoresis, Polyacrylamide Gel ; Glucosides ; Hydrogen-Ion Concentration ; Molecular Weight ; Oryza

This study examined the chemical composition of A. blasiliensis and the chemical structural properties of an immuno-stimulating polysaccharide. The amino acids, free sugars, and organic acids by HPLC and fatty acids by GC were analyzed. The immuno-stimulating substance from A. blasiliensis was extracted with hot water and purified by ethanol precipitation. It underwent ion exchange chromatography on DEAE-cellulose and gel filtration on Toyopearl HW 65F. Through GP-HPLC, the substance was found to be homogeneous. Its chemical structure was determined by 13C-NMR. Fatty acids, organic acids, and sugar alcohol composition consisted exclusively of linoleic acid, fumaric acid and mannitol, respectively. The amino acids were mainly glutamic acid, glycine, and arginine. By 13C-NMR analysis, the immuno-stimulating substance was identified as beta-(1-->3) (1-->6)-glucan, composed of a backbone with (1-->3)-linked D-glucopyranosyl residues branching a (1-->6)-linked D-glucopyranosyl residue. The beta-glucan from A. blasiliensis showed pronounced immuno-stimulating activity on the antibody-production ability of B-lymphocytes by the hemolytic suspension assay. In these results, A. blasiliensis was estimated to have potent pharmacological properties and potential nutritional values.
Agaricus ; Amino Acids ; Arginine ; B-Lymphocytes ; Carbohydrates ; Chromatography, Gel ; Chromatography, High Pressure Liquid ; Chromatography, Ion Exchange ; DEAE-Cellulose ; Ethanol ; Fatty Acids ; Fumarates ; Glutamic Acid ; Glycine ; Linoleic Acid ; Mannitol ; Nutritive Value ; Water

Cytochrome P-450 (CP-450) is one of the three components of the liver microsomal enzyme system which hydroxylates fatty acids, hydrocarbons and a variety of drugs and other foreign compounds. Female Balb/c mice were immunized with purified polychlorinated biphenyl (PCB) – induced CP-450 LMII. The spleen cells derived from immunized mice were fused with SP2 myeloma cells using polyethylene glycol (PEG 3500). The hybrid cells were selected by hypoxanthine-aminopterin-thymidine (HAT) medium and the culture fluid were screened by enzyme-linked immunosorbent assay to CP450 LMII. The hybrid cells(×107) were inoculated into intraperitoneal cavity of Balb/c mice for the purpose of production of ascetic fluids. Monoclonal antibody (Mab) was purified from ascitic fluid by DEAE cellulose ion exchange chromatography and I¹²⁵ labeled Mab was also confirmed by autoradiography and SDS-polyacrylamide gel electrophoresis (MW:55,000 and 110,000)
Animals ; Ascitic Fluid ; Autoradiography ; Chromatography, Ion Exchange ; Cytochrome P-450 Enzyme System* ; Cytochromes* ; DEAE-Cellulose ; Electrophoresis ; Enzyme-Linked Immunosorbent Assay ; Fatty Acids ; Female ; Humans ; Hybrid Cells ; Hydrocarbons ; Liver* ; Mice ; Polyethylene Glycols ; Rats* ; Spleen

Acetic Acid ; analysis ; Chromatography, Ion Exchange ; Workplace

Air ; analysis ; Anions ; analysis ; Chromatography, Ion Exchange ; Ions ; analysis ; Workplace

PURPOSE: To elucidate the anti-oxidant effect of extract fractions from Xanthium strumarium L. on lens protein by crosslinking assay. METHODS: [(1 4)C] N-formyl-lysine was synthesized and purified by ion exchange chromatography. The crosslinking activities of extract fractions(Xan Crude, Xan CHCl3, Xan EtAc and Xan H2O) to lens protein were determined by incorporation with [(14)C] N-formyl-lysine. RESULTS: It was observed that Xan Crude, Xan CHCl3, and Xan EtAc extracted from Xanthium strumarium L. showed approximately 10% of antioxidant effect whereas Xan H2O showed no effect by crosslinking assay. CONCLUSIONS: This study showed that the crosslinking assay described in this study can be developed as a potential tool to screen the anti-oxidant effect rapidly and accurately compared to MTT assay. The result was compared to MTT assay using Human Lens epithelial cell line.
Antioxidants ; Cataract ; Chromatography, Ion Exchange ; Epithelial Cells ; Humans ; Xanthium*

Chromatography, Ion Exchange ; Hemoglobins, Abnormal ; analysis ; Humans ; Male

Chromatography, Ion Exchange ; Hemoglobins, Abnormal ; analysis ; Humans ; Male

OBJECTIVETo develop a method for determination of mandelic acid (MA) and phenylglyoxylic acid (PGA) in urine by reagent-free ion chromatography.

METHODSIon chromatography was performed on an AS19 column with a gradient elution solution containing 10-35 mmoL/L KOH at a flow rate of 1.00 ml/min, and MA and PGA were detected at ultraviolet wavelengths of 225 nm and 254 nm, respectively. The samples were diluted 10 times with purified water, then purified on a silver column to remove high concentrations of chloride ion, and injected after being filtered through a 0.2-µm m filter membrane.

RESULTSThe recoveries of standard addition of MA and PGA were 96.5% and 99.3%, respectively, with both relative standard deviations less than 5.0%. Good linear relationships were noted in the range of 1.0-100.0 mg/L for both MA and PGA (r >0.9995). The detection limits of MA and PGA were 0.02 mg/L and 0.05 mg/L, respectively; the minimum detectable concentrations of MA and PGA were 0.2 mg/L and 0.5 mg/L (when the sampling amount was 5.0 ml and diluted to 50.0 ml with water, and the injection volume was 300 µL).

CONCLUSIONSThis method is fast, convenient, and highly sensitive and selective. It can be used for the analysis of MA and PGA in the urine of styrene-exposed workers.

A bromoperoxidase from Gracilaria lemaneiformis was purified to homogeneity using a multi-step process of ammonium sulfate precipitation (AS), dialysis, and DEAE-cellulose 52 anion exchange chromatography. The bromoperoxidase activity was unstable or undetectable in crude extract solution. However, it became stable with electrophoretic purity after this multiple purification process. The anion exchange chromatography purification was a critical step in the purification process, which effectively eliminated the phycobiliprotein and smucilaginous polysaccharides. The purified bromoperoxidase was a monomeric enzyme with the relative molecular masses of 66 kD as determined by denaturing and native gradient gel electrophoresis. The optimal pH for bromoination was 6.0 and bromoperoxidase activity was stable as stored at a broad pH range of 3.0-9.0. Of a range of compounds tested, only vanadium enhanced bromoperoxidase activity. Kinetic studies for the bromination of monochlorodimedone (MCD) showed that the Km values of Br- and H2O2 are 53.5 micromol/L, 38 micromol/L respectively.
Chromatography, Ion Exchange ; methods ; Enzyme Stability ; Gracilaria ; enzymology ; Hydrogen-Ion Concentration ; Kinetics ; Peroxidases ; isolation & purification ; metabolism