János Holló

Interaction of taxol and other anticancer drugs with hydroxypropyl-β-cyclodextrin

Abstract The interaction between 23 anticancer drugs and hydroxypropyl-β-cyclodextrin (HPβCD) was studied by reversed-phase charge-transfer thin-layer chromatography and the relative strength of interaction was calculated. HPβCD formed inclusion complexes with 15 compounds, the complex always being more hydrophilic than the uncomplexed drug. The inclusion forming capacity of drugs differed considerably according to their chemical structure. The intensity of interaction significantly increased with increasing hydrophobicity of the guest molecule, demonstrating the preponderant role of hydrophobic interactions in inclusion complex formation.

Comparison of the kinetics of acetate biomethanation by raw and granular sludges

SummaryThe specific gas production rate for various anaerobic sludges was determined as a function of acetate concentration at 35°C and pH 7.0. Three substrate inhibition kinetic equations were fitted to experimental data by a non-linear regression method. The best description was reached with modified Haldane-type kinetics. Comparison of raw and granular sludges of different origin showed reduced sensitivity of granular sludges to substrate inhibition. A continuous experiment with molasses waste-water in an upflow anaerobic sludge blanket reactor gave similar results: substrate inhibition continuously decreased as granule development proceeded. by modification of the model real physical meaning was attributed to calculated kinetic parameters. This allowed more adequate comparison with data reported previously based on the equations of Monod and Andrews. Our results are of the same magnitude as previously reported data.

A study by means of lactone inhibition of the role of a “half-chair” glycosyl conformation at the active centre of amylolytic enzymes

Abstract The kinetics of inhibition of porcine-pancreatic alpha amylase, sweet-potato beta amylase, and Aspergillus niger glucamylase enzymes have been studied by use of d -glucono-l,5-lactone and maltobiono-1,5-lactone as transition-state analogs. With d -glucono-1,5-lactone, alpha amylase can be inhibited, to a degree, non-competitively (K i 0.81m M ,β≈e0.2), whereas with maltobionolactone, the inhibition is competitive (K i 10.31m M ). The effect of beta amylase can be inhibited with maltobionolactone in a completely competitive way (K i O.11m M ), whereas with d -gluconolactone the inhibition is very poor (K i 21m M ). Glucoamylase cannot be inhibited with maltobionolactone, whereas with d -gluconic acid, a completely mixed inhibition way be observed (K i 1.3m M ). The ratio of the binding affinity of the lactones, products, and substrates, permits the conclusion that ring distortion takes place in the transition state with all three enzymes.

A study of the role of histidine side-chains at the active centre of amylolytic enzymes☆

Abstract The role of histidine side-chains in reactions catalysed by porcine-pancreatic alpha-amylase, sweet-potato beta-amylase, and Aspergillus niger glucamylase has been studied by using diethyl pyrocarbonate, a specific protein reagent. Changes in the activity, binding affinity, and apparent kinetic parameters due to ethoxycarbonylation have been determined. For pancreas alpha-amylase, four of the eight histidine groups, for sweet-potato beta-amylase, six of the seven histidine groups, and for A. niger glucamylase, four of the six histidine groups were shown to be ethoxycarbonylated. Ethoxycarbonylation occurred as an apparent first-order reaction, with rate constants in the range 3.6–4.9 x 10−2min−1. Ethoxycarbonylation of the histidine group at the active centre rapidly inactivated alpha-amylase, whereas the other three groups are not located in the active centre, although activity and substrate binding are only slightly affected by their modification. For beta-amylase and glucamylase, only slight or no change in activity could be detected on ethoxycarbonylation, whereas significant changes were observed in the binding affinity.

Interaction of taxol and other anticancer drugs with α-cyclodextrin☆

Abstract The interaction between 23 anticancer drugs and α-cyclodextrin (α-CD) was studied by reversed-phase charge-transrer thin-layer chromatography and the relative strength of interaction was calculated. As α-CD has smaller cavity than β- and τ-CD it interacted only with 10 anticancer drugs proving the relatively poor complex forming capacity of α-CD. The hydrophobicity of host-guest inclusion complex was always different from that of the uncomplexed drug suggesting that the complex formation may influence the uptake, absorption, half-life etc. of the original drug. the inclusion forming capacity of drugs differed considerably according to their chemical structure. The intensity of interaction significantly depended on the hydrophobicity of the guest molecule proving the preponderant role of hydrophobic interactions in inclusion complex formation.

The influence of organic load on granular sludge development in an acetate-fed system

SummaryThe development of granular sludge in laboratory-scale upflow anaerobic sludge-blanket reactors was studied. Acetate was supplied as sole carbon source in order to select the acetotrophs Methanosarcina and Methanothrix. These microorganisms are dominant in methanogenic ecosystems and their ratio seems to control the speed of granulation. Changing the ratio of the above species was followed on the basis of their different F420-coenzyme content. Five reactors were operated at the same hydraulic retention time but at different feed substrate concentrations. We found that granulation takes place only in acetate-fed systems but this process was slower and the resultant granules looser and less stable than those developed on sugar-starch substrate. In the range of feed acetate levels examined (0.5−0.3 g/1) higher concentrations of feed caused faster granulation of the sludge bed and, presumably, of the microbial population, and resulted in larger granules containing sludge that settled more readily. We found no evidence for selection pressure at substrate concentrations below 0.5 g/1 acetate in the reactor.

A study of the role of tyrosine groups at the active centre of amylolytic enzymes

Abstract The role of exposed tyrosine side-chains in enzyme-catalysed reactions has been studied for porcine-pancreatic alpha-amylase, sweet-potato beta-amylase, and Aspergillus niger glucamylase using N -acetylimidazole as the specific protein reagent. The changes in activity binding affinity (Δ k −1 / k +1 ), and kinetic parameters ( K m, k 2 ) due to acetylation of the phenolic hydroxyl groups have been determined. Acetylation of each enzyme occurred by an “apparent” first-order reaction with a rate constant of 0.72–1.4 x 10 −1 min −1 . Acetylation increased the apparent K m (soluble starch as the substrate) for each enzyme (appreciably for alpha-amylase and glucamylase), whereas k 2 remained unchanged. Similarly, for each enzyme, the binding affinity for immobilised cyclohexa-amylose decreased appreciably, whereas the catalytic activity was reduced only to a small degree (and remained unchanged for beta-amylase). It is concluded that the tyrosine groups located in the active centre of each enzyme have a substrate-binding function.

New interpretation of expansion in biofilm-coated particle fluidization

Biofilm processes are widely used; the most effective of these fluidized bed reactors, the design and modelling of which is still difficult. A fluidized sand bed denitrifying reactor was run to clarify contradictions in the applicability of the Richardson-Zaki model for the description of fluidization hydrodynamics and to determine fully the true biofilm parameters experimentally. Biofilm density was found to be 1.055 ±0.018 g/cm3. A fundamental parameter of the Richardson-Zaki model, the terminal settling Reynolds number, was found to be not characteristic of the particles. Consequently a new approach was developed for the description of biofilm-coated particle fluidization. The model is based on two new parameters: the ‘expansion coefficient’ and the ‘specific occupied particle volume at zero flow’, which are readily determinable and characteristic parameters of the fluidized particles, being independent of reactor size and shape, liquid velocity or of the quantity of carrier particles. The model is suitable for modelling bed porosity or biomass concentration as a function or the biofilm thickness and upflow liquid velocity. We found that there can be an optimal biofilm thickness above which not only can the diffusion limitation increase, but the overall biomass concentration decreases at a given liquid velocity.

The biosynthesis of starch : Part XIII. Inhibition of potato phosphorylase by donor substrate analogues☆☆☆

Abstract The inhibitory effect of d -glucose, 2-deoxy- d - arabino -hexose, 3- O -methyl- d -glucose, and 6- O -methyl- d -glucose on the enzymic synthesis catalysed by potato phosphorylase has been investigated. Kinetic methods showed that, for both of the substrates employed, a competitive inhibitor effect was exerted by each of the substrate analogues examined. Values for K i and K m calculated from the experimental data indicate that, in the presence of each of the added substrate analogues, the affinity of the enzyme for both substrates decreased. On the basis of the changes in K i values, it appeared that, in the case of each substrate, the inhibitor effect of d -glucose was suppressed by the absence of the hydroxyl groups at the positions 2 and 3.

A pressure test method monitors biofilm coated particle fluidization

A fluidized bed denitrifying reactor was run to examine the vertical segregation of sand particles on the basis of different biofilm coverage, so far neglected when modelling fluidized beds. The segregation was found to be significant and it can be directly correlated with the vertical hydrostatic pressure profile in the bed. A procedure was developed for the rapid determination of biofilm thickness from hydrostatic pressure data using a recently published method based on the use of the novel criteria “expansion coefficient” and “specific occupied volume”. A key feature of the procedure is the “particle content”, which can be calculated from particle characteristics and is correlated in this study with the hydrostatic pressure gradient. The method was verified by directly measuring biofilm thickness as a function of the vertical position in the bed. This way biofilm thickness can be calculated from a readily measurable hydrostatic pressure profile with an error of 0.04–0.06 mm. This error is believed to be due to N2 gas entrapment in the denitrifying biofilm and to the original inaccuracy of the determination of particle size and volume. The method is rather insensitive to the exact biofilm density when the usual high-density carrier material is used.