Glucuronic acid directly linked to galacturonic acid in the rhamnogalacturonan backbone of beet pectins.

Sugar-beet pulp was de-esterified and submitted to 72 h hydrolysis by 0.1 M HCl at 80 degrees C. Oligomers containing a single glucuronic acid (GlcA) moiety in addition to n(>/= 2) repeats of the dimer -->4)-alpha-D-GalpA-(1-->2)-alpha-L-Rhap-(1--> were isolated from the hydrolysate by ion-exchange and gel-permeation. Glycosyl linkage composition analysis and 1H NMR studies indicated that the GlcA was attached to O-3 of a galacturonic acid (GalA) residue, as shown for the two pentamers beta-D-GlcpA-(1-->3)-alpha-D-GalpA-(1-->2)-alpha-L-Rhap-(1-->4)-alpha-D-GalpA-(1-->2)-L-Rhap and alpha-D-GalpA-(1-->2)-alpha-L-Rhap-(1-->4)-[beta-D-GlcpA-(1-->3)]-alpha-D-GalpA-(1-->2)-L-Rhap. Substitution by GlcA was estimated as occurring on one GalA residue out of 72 in the rhamnogalacturonan fraction of the backbone of beet pectins.     

A single-sample method for determination of carbohydrate and protein contents glycoprotein bands separated by sodium dodecyl sulfate- polyacrylamide gel electrophoresis.

A method is described for determination of carbohydrate and protein contents of glycoproteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then electroblotted onto polyvinylidene difluoride (PVDF) membranes. Blots were stained, and appropriate pieces of PVDF membranes were excised, destained, and subjected to sequential hydrolysis with 0.2 M trifluoroacetic acid (TFA) for 1 h at 80 degrees C, then with 2 M TFA for 4 h at 100 degrees C, and finally with 6 M HCl at 100 degrees C for 24 h to release sialic acids, neutral sugars with hexosamines, and amino acids, respectively. In some instances preliminary methanolysis was used. Carbohydrates including sialic acids were quantitated by high pH anion exchange chromatography with pulsed amperometric detection. Protein content of the bands was determined as amino acids by the fluorescamine or ninhydrin method. In the calculation of results proper adjustments were made for small amounts of fucose released by hydrolysis with 0.2 M TFA at 80 degrees C, and for partial degradation of protein during hydrolysis with 2 M TFA at 100 degrees C. Recoveries of amino acids from hydrolysates of glycoproteins that had been electroblotted onto PVDF membranes equaled those of carbohydrates. This was possible because of preliminary hydrolysis of glycoproteins with TFA, as well as washing of wet, instead of dried, PVDF membranes after hydrolysis with 6 M HCl. The two modifications increased yields of amino acids by about 30%. The method was successfully applied to the determination of molar and weight percentage composition of human transferrin, band 3 protein, glycophorin A, and alpha(1)-acid glycoprotein. In each case the results obtained for directly hydrolyzed and electrophoresed/electroblotted glycoproteins were practically identical. We also determined the glucosamine content of band 4.1 protein of erythrocytes.     

The reaction of hyaluronic acid and its monomers, glucuronic acid and N-acetylglucosamine, with reactive oxygen species

Synovial fluid is a approximately 0.15% (w/v) aqueous solution of hyaluronic acid (HA), a polysaccharide consisting of alternating units of GlcA and GlcNAc. In synovial fluid of patients suffering from rheumatoid arthritis, HA is thought to be degraded either by radicals generated by Fenton chemistry (Fe2+/H2O2) or by NaOCl generated by myeloperoxidase. We investigated the course of model reactions of these two reactants in physiological buffer with HA, and with the corresponding monomers GlcA and GlcNAc. meso-Tartaric acid, arabinuronic acid, arabinaric acid and glucaric acid were identified by GC-MS as oxidation products of glucuronic acid. When GlcNAc was oxidised, erythronic acid, arabinonic acid, 2-acetamido-2-deoxy-gluconic acid, glyceric acid, erythrose and arabinose were formed. NaOCl oxidation of HA yielded meso-tartaric acid; in addition, arabinaric acid and glucaric acid were obtained by oxidation with Fe2+/H2O2. These results indicate that oxidative degradation of HA proceeds primarily at glucuronic acid residues. meso-Tartaric acid may be a useful biomarker of hyaluronate oxidation since it is produced by both NaOCl and Fenton chemistry.     

Depolymerization and de-N-acetylation of chitin oligomers in hydrochloric acid

The monosaccharide 2-amino-2-deoxy-D-glucose (glucosamine, GlcN) has recently drawn much attention in relation to its use to treat or prevent osteoarthritis in humans. Glucosamine is prepared from chitin, a process that is performed in concentrated acid, such as hydrochloric acid. This process involves two acid-catalyzed processes, that is, the hydrolysis of the glycosidic linkages (depolymerization) and of the N-acetyl linkages (de-N-acetylation). The depolymerization reaction has previously been found to be much faster compared to the deacetylation, with the consequence that the chitin chain will first be hydrolyzed to the monomer 2-acetamido-2-deoxy-D-glucose (N-acetylglucosamine, GlcNAc) which is subsequently deacetylated. We have found that the chitin disaccharide GlcNAc(1-->4)GlcNAc could be completely hydrolyzed to the monosaccharide GlcNAc with negligible concomitant de-N-acetylation, and the chitin disaccharide and monosaccharide were further used to study the depolymerization reaction and the de-N-acetylation reaction, respectively. The reactions were performed in hydrochloric acid as a function of acid concentration (3-12 M) and temperature (20-35 degrees C), and 1H-NMR spectroscopy was used to monitor the reaction rates. The 1H NMR spectrum of GlcNAc in concentrated (12 M) and deuterated hydrochloric acid at 25 degrees C was assigned. The glucofuranosyl oxazolinium (3) ion was found to exist in equilibrium with the alpha- and beta-anomers of the pyranose form of GlcNAc, where 3 was present in half the total molar concentrations of the two anomeric forms of GlcNAc. At lower acid concentration (3-6 M), only trace concentrations of 3 could be detected. The rate of de-N-acetylation of GlcNAc was determined as a function of hydrochloric acid concentration, showing a maximum at 6 M and decreasing by a factor of 2 upon decreasing or increasing the acid concentration to 3 or 12 M. The activation energy for hydrolysis of the N-acetyl linkage of GlcNAc was determined to be 102 +/- 7, 116 +/- 8, and 110 +/- 8 kJ mol(-1) at 3, 6, and 12 M hydrochloric acid concentration, respectively. The results are in accordance with the proposed SN2 reaction mechanism of the acid-catalyzed hydrolysis of the N-acetyl linkage where the rate-limiting step is the addition of water to the carbonium ion. The 1H NMR spectrum of the dimer GlcNAc-GlcNAc in concentrated (12 M) and deuterated hydrochloric acid at 25 degrees C was assigned. The rate of the acid-catalyzed cleavage of the glycosidic linkage of the dimer was determined as a function of hydrochloric acid concentration, showing a 6-fold increase from 3 to 6 M HCl concentration and a further 6-fold increase from 6 to 12 M HCl concentration, in contrast to the much smaller effect of acid concentration on the deacetylation reaction. Activation energy for hydrolysis of the glycosidic linkage of GlcNAc-GlcNAc was determined to be 110 +/- 6, 111 +/- 6, and 112 +/- 4 kJ mol(-1) at 3, 6 and 12 M hydrochloric acid concentration, respectively, that is, very similar to the activation energies determined for the deacetylation reaction. The results are in accordance with the proposed SN1 reaction mechanism of the acid-catalyzed hydrolysis of the glycosidic linkage, where the rate-limiting step is the formation of the carbonium ion.     

Studies on the chemical structure of the core-lipid A region of the lipopolysaccharide of Acinetobacter calcoaceticus NCTC 10305. Detection of a new 2-octulosonic acid interlinking the core oligosaccharide and lipid A component

Lipopolysaccharide of Acinetobacter calcoaceticus NCTC 10305 was treated with acid (0.1 M HCl, 100 degrees C, 1 h). The product obtained (LPSdegr) was subjected to various modification and degradation procedures including reduction, hydrazinolysis and strong acid hydrolysis. Methylation analysis of purified part structures revealed the presence of a 4'-phosphorylated (beta 1'-6)-linked D-glucosamine disaccharide (lipid A backbone), which carried in position 6' a hitherto unknown 2-octulosonic acid (OclA) in highly acid-stable linkage. It was further shown that OclA is substituted in position 5 by a glucose tetramer, the reducing residue of which is phosphorylated. The hydrophilic region of the LPSdegr could thus be characterized as a phosphorylated heptasaccharide of the following structure: (Formula: see text).     

链球菌生物合成透明质酸的分子机理与基因工程菌构建进展

透明质酸（Hyaluronan, HA）是由葡萄糖醛酸和N-乙酰氨基葡萄糖为双糖单位交替连接而成的粘多糖物质。目前构建基因工程菌成为提高产量和改善品质的重要手段。本文从发酵菌种、操纵子、关键酶和工程菌构建等方面,综述了链球菌HA生物合成的分子机理,分析了当前生产中存在的问题,并提出了解决问题的方法。     

Phosphoamino acid analysis of protein immobilized on polyvinylidene difluoride membrane

The direct analysis of phosphorylated proteins bound to polyvinylidene difluoride membrane (PVDFm) has been examined. Use of 14C-methylated marker proteins demonstrated that proteins electroblotted on PVDFm were quantitatively retained through a series of test conditions, which included 1 M hydroxylamine (25 degrees C, 30 min), 0.1 M NaOH (37 degrees C, 30 min), 0.1 M HCl (55 degrees C, 2 h), and 6 U/ml alkaline phosphatase (pH 9.5, 37 degrees C, 24 h). Approximately half the protein remained bound following 2-h treatment in 1 M KOH (55 degrees C). The same series of test conditions were employed to assess the stability of phosphorylated residues in 32P-labeled protein immobilized on PVDFm, in order to assign them as carboxyl-,N-, or O-linked groups. The properties of phosphorylated proteins as determined by this method were comparable to the properties that have been reported for soluble proteins. Use of the PVDFm immobilization step affords simplification of the experimental procedures and permits rapid, quantitative sample recovery using submicrogram quantities of protein. Further, the PVDFm-bound phosphoproteins could be subjected to partial acid hydrolysis directly on the membrane and required no further purification for subsequent identification of the labeled phosphohydroxyamino acids. Definitive identification of labeled phosphoserine residues in histone, phosphoserine and phosphothreonine residues in myelin basic protein and insulin receptor, and phosphotyrosine residues in autophosphorylated insulin receptor was accomplished with as little as 0.2 nCi in about 50 ng of phosphorylated protein.     

Different instability of nuclear DNA at acid hydrolysis in cancerous and noncancerous cells as revealed by fluorescent staining with acridine orange

Ehrlich cancer cells and inflammatory cells in mouse ascitic fluid were hydrolyzed and stained with acridine orange (AO). The AO hydrolysis curves for G1/G2 + M phase cancer cells and inflammatory cells were differentially determined using flow cytometry by monitoring the metachromatic red-shifted fluorescence of the fluorochrome bound to the single-stranded DNA produced by acid hydrolysis. By computer fitting of the Bateman function to the hydrolysis curves, the kinetic parameters k1 (rate constant for the production of single-stranded DNA), k2 (rate constant for the degradation of the produced single-stranded DNA), and y0 (theoretical value of the single-stranded DNA present initially) were determined. It was found that the k2 value, which reflects the degree of DNA instability, was much higher for cancer cells in both the G1 and G2 + M phases than for inflammatory cells. This finding led us to develop a method for the differential AO staining of cancer cells and non-cancerous cells utilizing the different degree of DNA instability at acid hydrolysis. AO staining after hydrolysis with 2N HCl at 30 degrees C for 8.5 min was found to be the optimal method. In the 60 cases of human malignant epithelial and nonepithelial tumors tested, all of the malignant tumor cells emitted metachromatic red fluorescence, while all of the nonmalignant tumor cells (5 cases of benign tumor) and normal cells emitted orthochromatic green fluorescence when observed with a violet excitation light under a fluorescence microscope. This new technique can be a useful tool for the screening of malignancy in exfoliative cytology and also for basic cancer research.     

Chemical stability of prostacyclin (PGI2) in aqueous solutions.

The rate constant for the hydrolysis of prostacyclin (PGI2) to 6-keto-PGF1alpha was measured by monitoring the UV spectral change, over a pH range 6 to 10 at 25 degrees C and the total ionic strength of 0.5 M. The first-order rate constant (kdegreesobs) extrapolated to zero buffer concentration follows an expression, kdegreesobs = kH+ (H+), where kH+ is a second-order rate constant for the specific acid catalyzed hydrolysis. The value of kH+ obtained (3.71 x 10(4) sec-1 M-1) Is estimated approximately 700-fold greater than a kH+ value expected from the hydrolysis of other vinyl ethers. Such an unusually high reactivity of PGI2 even for a vinyl ether is attributed to a possible ring strain release that would occur upon the rate controlling protonation of C5. A Br?nsted slope (alpha) of 0.71 was obtained for the acid (including H3O+) catalytic constants, from which a pH independent first-order rate constant for the spontaneous hydrolysis (catalyzed by H2O as a general acid) was estimated to be 1.3 x 10(-6) sec-1. An apparent activation energy (Ea) of 11.85 Kcal/mole was obtained for the hydrolysis at pH 7.48, from which a half-life of PGI2 at 4 degrees C was estimated to be approximately 14.5 min. when the total phosphate concentration is 0.165 M (cf. 3.5 min. at 25 degrees C).     

1H NMR of glycosaminoglycans and hyaluronic acid oligosaccharides in aqueous solution: the amide proton environment

The exchangeable amide protons of hyaluronic acid (HA) oligosaccharides and a higher-molecular-weight segment dissolved in H2O at pH 2.5 or 5.5 were examined by H NMR spectroscopy at 250 MHz. The HA segment preparation showed a single amide resonance, near the chemical shift for the amide proton of the monosaccharide 2-acetamido-2-deoxy-beta-D-glucopyranose (beta-GlcNAc). Smaller HA oligosaccharides showed two or three separate amide proton resonances, corresponding in relative peak area to interior or end GlcNAc residues. The interior GlcNAc amide resonance occurred at the same chemical shift as the single resonance of the HA segment. For the end GlcNAc residues, linkage to D-glucuronopyranose (GlcUA) through C1 resulted in an upfield shift relative to the beta-anomer of GlcNAc, whereas linkage through C3 resulted in a downfield shift relative to the corresponding anomer of GlcNAc. These chemical-shift perturbations appeared to be approximately offsetting in the case of linkage at both positions. The amide proton vicinal coupling constant (ca. 9 Hz) was found to be essentially independent of chain length, residue position, or solution pH. These data favor a nearly perpendicular orientation for the acetamido group with respect to the sugar ring, little affected by linkage of GlcNAc to GlcUA. No evidence for the existence of a stable hydrogen bond linking the amide proton with the carboxyl(ate) oxygen of the adjacent uronic acid residue was found. The amide proton resonances for chondroitin, chondroitin 4-sulfate, and dermatan sulfate were compared to that of HA. The chemical shifts of these resonances deviated no more than 0.1 ppm from that of HA. A small dependence on the identity of the adjacent uronic acid residue was noted, based on the observation of two resonances for dermatan sulfate.     

Stability of glucose oxidase and catalase adsorbed on variously activated 13X zeolite

The use of 13X zeolite (0.1-0.4-mm granules), treated with 2N and 0.01N HCI, 0.01M citric acid, 0.1M citric-phosphate buffer (pH 3.6), and in untreated form to adsorb glucose oxidase of fungal origin and microbial catalase was examined. Physicochemical analysis of the support demonstrated that its crystalline structure, greatly altered by the HCl and buffer, could be partially maintained with citric acid. The specific adsorption of the enzymes increased with decreasing pH and proved to be considerable for all the supports. The stability with storage at 25 degrees C is strictly correlated with the titrable acidity of the activated zeolite expressed as meq NaOH/g and with pH value of the activation solution. It proved to be lower than 55 h for both enzymes if adsorbed on zeolite treated with 2N HCl, and 15-fold and 30-fold higher for glucose oxidase and catalase adsorbed, respectively, on zeolite treated with the 0.1M citric-phosphate buffer and 0.01M citric acid. The specific adsorption of glucose oxidase and catalase was, respectively, 1840 U/g at pH 3.0 and 6910 U/g at pH 5.0. Their half-life at 25 degrees C with storage at pH 3.5 for the former and at pH 5.0 for the latter was 800 and 1560 h vs. 40 and 110 h for the corresponding free enzymes.     

2-O-(beta-D-glucuronyl)-7-O-(2-amino-2-deoxy-alpha-D-glucopyranosyl)-L-glycero-D-manno-heptose: a constituent of the Bordetella pertussis endotoxin.

The presence of bound D-glucuronic acid in the endotoxin of Bordetella pertussis was demonstrated. The branched chain trisaccharide named in the title was isolated after hydrolysis of the endotoxin with 3 M HCl for 2 h at 100 degrees C. Its structure was established by chemical and enzymic degradation.     

Recovery of tryptophan in peptides and proteins by high-temperature and short-term acid hydrolysis in the presence of phenol

The addition of 3% (w/v) phenol to 6 M HCl largely prevented the destruction of tryptophan during rapid hydrolysis of peptides and proteins at 166 degrees C for 25 min or at 145 degrees C for 4 h. This hydrolysis procedure was advantageous for amino acid microanalysis using conventional high-performance liquid chromatography with a precolumn derivatization technique. The recovery of tryptophan from proteins was at least 80%. The addition of phenol also improved the recovery of methionine and carboxymethylcysteine. The amount of tryptophan in proteins electroblotted onto a polyvinylidene difluoride membrane was determined by this method.     

Isolation of a trisaccharide containing 2-amino-2-deoxy-D-galacturonic acid from the Bordetella pertussis endotoxin

4-O-(2-Amino-2-deoxy-alpha-D-glucopyranosyl-6-O-(2-amino-2-deoxy-alpha-D-galactopyranuronyl)-D-glucopyranose, a branched-chain trisaccharide, was isolated after hydrolysis of Bordetella pertussis endotoxin with 4 M HCl for 1 h at 100 degrees C. The trisaccharide was present in both polysaccharide moieties of the two constituent lipopolysaccharides of this endotoxin. Its structure was established by analysis of the 400-MHz nuclear magnetic resonance spectrum and by chemical and enzymatic degradation.     

Production and characterization of a thermostable chitinase from a new alkalophilic Bacillus sp. BG-11

An alkalophilic, environmental micro-organism, Bacillus sp. BG-11, has been isolated and characterized. It produced 76 U ml-1 of chitinase in liquid batch fermentation after 72 h of incubation at 50 degrees C using chitin-enriched medium. The molecular weight of purified chitinase was estimated to be 41 kDa by SDS-PAGE. The pH and temperature optima of chitinase immobilized on chitosan and calcium alginate were 8.5 and 50 degrees C, respectively, which were same as that of free enzyme. The pH and thermostability of immobilized chitinase were enhanced significantly. The chitinase immobilized on chitosan was stable between pH 5.0 and 10.0, and the half-life of chitosan-immobilized enzyme at 70, 80 and 90 degrees C was 90, 70 and 60 min, respectively. The end-products formed during the enzyme-substrate reaction were identified by 13C-NMR, and N-acetyl-D-glucosamine was found to be the major end-product. GlcNAc (GlcNAc)2 and (GlcNAc)3 inhibited the chitinase activity by 32, 25 and 18%, respectively, at a concentration of 10 mmol l-1. The shelf-life of chitinase (retained 100% activity) at 4 degrees C was 8 weeks in the presence of either sodium azide (100 microgram ml-1), sodium metabisulphite (0.1% w/v) or KCl (15% w/v). The enzyme was resistant to the action of proteases and allosamidin.     

Pre-steady-state and steady-state kinetic analysis of the low molecular weight phosphotyrosyl protein phosphatase from bovine heart.

The complete time course of the hydrolysis of p-nitrophenyl phosphate catalyzed by the low molecular weight (acid) phosphotyrosyl protein phosphatase from bovine heart was elucidated and analyzed in detail. Burst titration kinetics were demonstrated for the first time with this class of enzyme. At pH 7.0, 4.5 degrees C, a transient pre-steady-state "burst" of p-nitrophenol was formed with a rate constant of 48 s-1. The burst was effectively stoichiometric and corresponded to a single enzyme active site/molecule. The burst was followed by a slow steady-state turnover of the phosphoenzyme intermediate with a rate constant of 1.2 s-1. Product inhibition studies indicated an ordered uni-bi kinetic scheme for the hydrolysis. Partition experiments conducted for several substrates revealed a constant product ratio. Vmax was constant for these substrates, and the overall rate of hydrolysis was increased greatly in the presence of alcohol acceptors. An enzyme-catalyzed 18O exchange between inorganic phosphate and water was detected and occurred with kcat = 4.47 x 10(-3) s-1 at pH 5.0, 37 degrees C. These results were all consistent with the existence of a phosphoenzyme intermediate in the catalytic pathway and with the breakdown of the intermediate being the rate-limiting step. The true Michaelis binding constant Ks = 6.0 mM, the apparent Km = 0.38 mM, and the rate constants for phosphorylation (k2 = 540 s-1) and dephosphorylation (k3 = 36.5 s-1) were determined under steady-state conditions with p-nitrophenyl phosphate at pH 5.0 and 37 degrees C in the presence of phosphate acceptors. The energies of activation for the enzyme-catalyzed hydrolysis at pH 5.0 and 7.0 were 13.6 and 14.1 kcal/mol, respectively. The activation energy for the enzyme-catalyzed medium 18O exchange between phosphate and water was 20.2 kcal/mol. Using the available equilibrium and rate constants, an energetic diagram was constructed for the enzyme-catalyzed reaction.     

Purification and properties of pig brain guanine deaminase.

Guanine deaminase (guanine aminohydrolase, EC 3.5.4.3) from pig brain was purified to homogeneity by column chromatography and ammonium sulphate fractionation. Homogeneity was established by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulphate (SDS). The molecular weight of 110 000 was determined by gel filtration and sucrose density gradient centrifugation. SDS polyacrylamide gel electrophoresis indicated subunits of a molecular weight of 50 000. The amino acid composition, the isoelectric point and the number of -SH groups were determined. 5.5'-Dithiobis-(2-nitrobenzoic acid) reacts with about seven -SH groups in the native enzyme, but upon denaturation with SDS, 10 -SH groups react with this former reagent. Using electrolytic reduction, 44 half-cystines were determined in accordance with the number of cysteic acid residues determined by amino acid analysis after performic acid oxidation. The Km values determined for substrates of the enzyme were 1.1 . 10(-5) M for guanine in 0.1 M Tris. HCl buffer (pH 8.0) and 3.3 . 10(-4) M for 8-azaguanine in 0.1 M phosphate buffer, pH 6.4. The pKa values determined for ionizable groups of the active site of the enzyme were near pH 6.2 and pH 8.2. The chemical and kinetic evidence suggests that cysteine and histidine may be essential for the catalysis.     

Purification and characterization of a novel isozyme of chitinase from Bombyx mori

75-kDa chitinase, which showed potential as a biocontrol agent against Japanese pine sawyer, was characterized after purification from the integument of the fifth instar larvae of Bombyx mori by chromatography on diethylaminoethyl (DEAE)-Toyoperal 650 (M), hydroxylapatite, and Fractogel EMD DEAE 650 (M) columns. The optimum pH was 6.0 toward N-acetylchitopentaose (GlcNAc5) and 10 toward glycolchitin. The optimum temperature was 60 degrees C toward GlcNAc5 and 25 degrees C toward glycolchitn. The enzyme was stable at pH 7-10 and below 40 degrees C. Kinetic analysis and reaction-pattern analysis using glycolchitin and N-acetylchitooligosacchraides as substrates indicated that 75-kDa chitinase is an endo- or random-type hydrolytic enzyme to produce the beta anomeric product and that it prefers the longer N-acetylchitooligosaccharides, suggesting, together with the N-terminal amino acid sequence, that the 75-kDa chitinase belongs to family 18 of glycosyl hydrolases.     

Chemical change involved in the oxidative reductive depolymerization of hyaluronic acid

The oxidative reductive depolymerization (ORD) of hyaluronate has been investigated. A solution of hyaluronate (Mr 4.07 x 10(5] in phosphate buffer (pH 7.2) was incubated in the presence of Fe2+ for 24 h at 37 degrees C under an oxygen atmosphere to yield depolymerized hyaluronate (ORD fragments; an average Mr of 2,600). The ORD fragments contain 21 and 24% less hexosamine and uronic acid, respectively, but no olefinic linkage. They were exhaustively digested with chondroitinase AC-II. The resulting oligosaccharides and monosaccharides were separated by gel filtration and ion-exchange chromatography, and their structures were determined by proton and carbon-13 NMR, fast atom bombardment mass spectrometry, and chromatographic techniques combined with chemical modifications. The following structures derived from the reducing ends of the ORD fragments were identified: 4,5-unsaturated GlcA(beta 1----3)-N-acetyl-D-glucosaminic acid (where GlcA- represents glucuronosyl-) (21%), 4,5-unsaturated GlcA(beta 1----3)GlcNAc(beta 1----3)-D-arabo-pentauronic acid (24%), and N-acetyl-D-glucosamine (51%). The following structures derived from the nonreducing ends were identified: L-threo-tetro-dialdosyl-(1----3)GlcNAc (a tentative structure, 8%), N-acetylhyalobiuronic acid (20%), and N-acetyl-D-glucosamine (45%). The results indicate that the ORD reaction of hyaluronate proceeds essentially by random destruction of unit monosaccharides due to oxygen-derived free radicals, followed by secondary hydrolytic cleavage of the resulting unstable glycosidic substituents.     

Biotechnology for the production of nutraceuticals enriched in conjugated linoleic acid: II. Multiresponse kinetics of the hydrolysis of corn oil by a Pseudomonas sp. lipase immobilized in a hollow-fiber reactor

The hydrolysis of corn oil in the presence of a lipase from Pseudomonas sp. immobilized within the walls of a hollow-fiber reactor was studied at 30 degrees C. To assess the selectivity of this immobilized enzyme, the effluent concentrations of five different free fatty acids were measured using high-performance liquid chromatography (HPLC). Several rate expressions associated with a generic ping-pong bi-bi mechanism were used to fit the experimental data for this lipase-catalyzed reaction. A multiresponse nonlinear regression method was employed to determine the kinetic parameters associated with these rate expressions. Quasi-optimum operating conditions corresponded to 30 degrees C and a buffer pH value of 7.0. Under these conditions, the concentration of free linoleic acid (C18:2) (the fatty acid of primary interest) in the effluent oil stream for a fluid residence time of 6 h was approximately 0.5 M.