published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

Lysozyme is a natural antimicrobial agent that is effective against many food spoilage and pathogenic microorganisms by disintegrating their cell walls. Immobilization of lysozyme has attractive applications for use in the food industry: (1) The enzyme could be readily separated from treated foods and beverages and re-used while the foods could still be claimed additive-free; (2) It could impart stable antimicrobial capability to the surface of food packaging polymers. In this study, a novel method is described for the preparation of a highly active immobilized lysozyme system. The method addressed three key issues in the covalent attachment of a biological active protein to an insoluble support: 1.) The protein should be attached to the matrix by the fewest possible bonds to minimize conformational change; 2.) The binding site(s) on the enzyme to the supports should be located as far as practical from its active center and be nonessential for its tertiary structure; 3.) The binding method should minimize the steric interference between the support and the immobilized enzyme. Using polystyrene resin beads as support matrix, peptide spacers of various lengths composed of 6-aminocaproic acid were synthesized with the solid phase peptide synthesis method. Then the amino terminals of the spacers were derivatized with bromoacetyl bromide and coupled to the protein's only histidine residue (His-15) that is nonessential for its lytic activity. Immobilized lysozyme with a spacer composed of three 6-aminocaproic acid units displayed the best lytic result against lyophilized M. lysodeikticus cells: 2736 U/g resin with a protein load of 2.21 mg/ resin. Retained activity was 14.2% of that of the free enzyme. Preparations with longer spacers yielded higher protein load yet the retained activity remained at about 14% level. A control consisted of random coupling of lysozyme to polystyrene beads without spacer gave an activity of 158 U/G with a protein load of 1.24 mg/g resin and 1.4% of retained activity, Properties of the immobilized lysozyme system were studied, including stability, effect of pH, surface characteristics of the support. A kinetics study of the system using Eadie-Hofstee plot demonstrated strong external diffusion effects, which resulted in deviation from classic Michaelis-Menton kinetic behavior. / Graduation date: 2004

Pyridoxal phosphate is a coenzyme in about 50 known enzymatic reactions. A simple and accurate method for the determination of pyridoxal phosphate would be desirable because it could provide a means to assess the nutritional status of vitamin B₆ in the human. The cyanohydrin methods to determine pyridoxal phosphate appear to be simple and promising. Cyanohydrin methods have been devised by Bonavita and Scardi, and Bonavita, and applied to biological materials by Yamada et al. The cyanohydrin procedure of Yamada et al. was investigated. In this procedure, the pyridoxal phosphate and pyridoxal in a deproteinized sample are separated with the use of a column of SM-cellulose (1 gm., equilibrated with 0.01 N acetic acid). Pyridoxal phosphate is eluted from SM-cellulose with 0.01 N acetic acid, and pyridoxal is eluted with 0.1 M sodium phosphate buffer, pH 7.4. Pyridoxal phosphate and pyridoxal are converted to their respective cyanohydrin derivatives by reaction with potassium cyanide. These cyanohydrin derivatives are measured fluorometrically at their activation and fluorescence maxima. In preliminary studies on the procedure by Yamada et al., the activation and fluorescence spectra of the cyanohydrin derivatives of pyridoxal phosphate and pyridoxal were obtained to determine the appropriate activating and fluorescent wavelength settings to use for subsequent fluorometric analyses. Pyridoxal phosphate cyanohydrin at pH 3.8 in 0.2 M sodium phosphate buffer had an activation maximum at 325 mμ and a fluorescence maximum at 415 mμ; and pyridoxal cyanohydrin at pH 10 in 0.2 M sodium phosphate buffer had an activation maximum at 355 mμ and a fluorescence maximum at 435 mμ. To obtain maximum fluorescence of the cyanohydrin derivatives, pyridoxal phosphate had to be reacted with potassium cyanide at 50°C for 60 minutes, and pyridoxal had to be reacted for 150 minutes. Following these preliminary studies, the elution pattern of pyridoxal phosphate and pyridoxal from a column of SM-cellulose was investigated. Pyridoxal phosphate was eluted with 0.01 N acetic acid; and pyridoxal, with both 0.01 N acetic acid and 0.1 M sodium phosphate buffer, pH 7.4. The recovery of pyridoxal phosphate from SM-cellulose was 93.5% when pyridoxal phosphate alone was applied to the column, and that of pyridoxal was 108.8% when pyridoxal alone was applied. When a mixture of pyridoxal phosphate and pyridoxal was applied to SM-cellulose, the recovery of pyridoxal phosphate was 105.5% and that of pyridoxal was only 59.8%. When either standard alone was added to blood, the recovery of pyridoxal phosphate in blood from SM-cellulose was 85.0%, and that of pyridoxal was only 29.1%. When a mixture of pyridoxal phosphate and pyridoxal was added to blood, the recovery of pyridoxal phosphate in blood from SM-cellulose was 62.6%, and that of pyridoxal was 52.1%. This lower recovery of pyridoxal phosphate in blood was due mainly to the high readings of the blanks. This higher recovery of pyridoxal phosphate in blood may be explained by the low concentration of pyridoxal in the buffer fractions from a column of SM-cellulose to which a mixture of pyridoxal phosphate and pyridoxal had been applied that was used to calculate the recovery. Determining the recovery of standards added to the supernatant after the precipitation of the proteins in blood, rather than to the hemolyzed blood before precipitation, would indicate whether pyridoxal phosphate and pyridoxal were lost by adsorption on the protein precipitate. The modified procedure of Yamada et al. is not sensitive enough to determine the pyridoxal phosphate and pyridoxal content of human blood. / Graduation date: 1968

The aim of the work in my thesis was to establish the extent to which the pathways of carbohydrate oxidation, namely, glycolysis and the oxidative pentose phosphate pathway, are compartmented in non-photosynthetic cells, with particular reference to the extent and organisation of these pathways in leucoplasts and amyloplasts. I pursued this aim by developing a technique for the isolation of both intact leucoplasts and amyloplasts from three-day-old soybean suspension cultures. This technique involved the enzymic preparation of protoplasts, gentle mechanical lysis of the protoplasts to release intact organelles, the layering of the organelles onto a 16-60% (w/w) linear sucrose density gradient, followed by the centrifugation of the gradient to allow separation of the organelles according to their densities. I then fractionated the gradient and measured marker enzymes in each fraction to show firstly, that I had concentrated the different plastids in different locations on the gradient, and secondly that the separate plastid fractions were not significantly contaminated by other cell components. I then measured the activities of all the enzymes of glycolysis and the oxidative pentose phosphate pathway in the isolated plastids. The results indicated that the leucoplasts contained all the enzymes of glycolysis, with the possible exception of enolase, and all the enzymes of the oxidative pentose phosphate pathway. The amyloplasts contained all the enzymes of both pathways. I confirmed these results with data from latency and protection experiments. I used my results to estimate the relative activities of the enzymes of carbohydrate oxidation in the plastids and the cytosol of soybean suspension cultures. In order to complement the results of the enzyme distributions, I developed a second technique, involving the use of a 0-40% (w/w) linear Nycodenz density gradient in place of the sucrose gradient, for the preparation of pure, intact, functional leucoplasts. I fed specifically labelled [<SUP>14</SUP>C]glucose 6-phosphate to these leucoplasts. The pattern of specifically labelled [<SUP>14</SUP>C] released as CO<SUB>2</SUB> suggested that the oxidative pentose phosphate pathway was operating in the leucoplasts, with recycling of both hexose and triose phosphates, and that glycolysis was operating down to 3-phosphoglycerate.

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The allosteric phosphofructokinase from <i>E. coli</i> crystallized in the absence of ligands. X-ray diffraction data were collected to 2.4 aa, AA resolution and the structure was solved by the method of molecular replacement, using the model of the liganded R-state of the enzyme. The atomic model was refined by rigid body refinement and by the least-squares method of Hendrickson and Konnert. The final structure is compared to the high resolution model of the liganded, active form of the enzyme and to the low resolution structure of the inhibited phosphofructokinase from B. stearothermophilus. It is found that the quaternary structure of the unliganded model is more similar to the liganded, active form than to the inhibited T-state of B. stearothermophilus pfk. There are however considerable differences in the tertiary and quaternary structures, apparently resulting from ligand binding. These changes are not localised to the binding sites. The overall change, though to result mainly from the binding of the activators, is consistent with the closing of the active site observed in the structure of the active state. The ways in which the ligand binding could bring about these changes are considered. The possible effect of the changes on the enzyme activity are discussed. It is possible that the structure represents an inactive T-state, different from that in the presence of inhibitors. Whatever the exact interpretation it is clear that the structure shows considerable flexibility suggesting that the two-state allosteric model is an oversimplification.

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