Catalytic behaviors of enzymes attached to nanoparticles: the effect of particle mobility

Nanoparticles provide an ideal remedy to the usually contradictory issues encountered in the optimization of immobilized enzymes: minimum diffusional limitation, maximum surface area per unit mass, and high effective enzyme loading. In addition to the promising performance features, the unique solution behaviors of the nanoparticles also point to a transitional region between the heterogeneous (with immobilized enzymes) and homogeneous (with soluble free enzymes) catalysis. The particle mobility, which is related to particle size and solution viscosity through Stokes-Einstein equation, may impact the reaction kinetics according to the collision theory. The mobility-activity relationship was examined through experimental studies and theoretical modeling in the present work. Polystyrene particles with diameters ranging from 110-1000 nm were prepared. A model enzyme, alpha-chymotrypsin, was covalently attached to the nanoparticles up to 6.6 wt%. The collision theory model was found feasible in correlating the catalytic activities of particles to particle size and solution viscosity. Changes in the size of particles and the viscosity of reaction media, which all affect the mobility of the enzyme catalyst, evidently altered the intrinsic activity of the particle-attached enzyme. Compared to K(M), k(cat) appeared to be less sensitive to particle size and viscosity.     

Effect of viscosity on enzyme-ligand dissociation. II. Role of the microenvironment

A theoretical treatment, describing a novel viscosity effect on decomposition of enzyme-ligand complexes, recently appeared (Somogyi et al., 1978). From this approach emerged a mechanistic picture of the manner in which increased viscosity lowers the value of the decomposition rate constant. A refined version of this model is presented herein. The analysis is extended to the molecular microenvironment ultimately responsible for mediating the "viscosity effect." Consideration is given to two major factors: (1) the role of viscosity in attenuating the excess chemical energy and (2) the statistical features of the microviscosity. In view of spatiotemporal inhomogeneity in the liquid structure, the concept of averaged microviscosity is introduced to parametrize the enzyme-ligand recombination probability. Quantitative predictions are consistent with models of liquid structure and with results from enzyme studies. The "viscosity effect" may contribute to substrate compartmentation in organized multi-enzyme systems in vivo.     

Hydrolysis of wheat starch and its effect on the falling number procedure: mathematical model

A population balance model was developed for wheat starch hydrolysis to simulate the performance parameters of a viscosity-based device, known as the Falling Number instrument. The instrument is widely used as an indirect means to gauge the level of preharvest sprout activity in cereal grains such as wheat and barley. The model consists of three competing kinetics: starch gelatinization, enzymatic hydrolysis, and enzyme thermal deactivation. Using established principles of starch rheology and fluid mechanics, the model simulates the velocity profiles of the falling stirrer, starch gel viscosity, and the Falling Number readings at various levels of alpha-amylase. Model predictions for the velocity of the stirrer at any time during the downward fall, as well as the prediction of the total time needed for the fall, defined as the Falling Number, were in fair agreement with experimental measurements. There was better agreement between the modeled viscosity and the final viscosity of the starch gel as measured by a precision rheometer than there was with the measured Falling Number.     

Studies on the viscosity of solutions of bovine liver glutamate dehydrogenase and on related hydrodynamic models; effect of toluene on enzyme association

The viscosity of bovine liver glutamate dehydrogenase solutions was studied at 10 and 20° C in 0.2.M sodium phosphate buffer at pH 7, in the concentration range 0.1–8 mg/ml. A method for the study of the viscosity of very dilute solutions of associating enzymes is described. It was found that the reduced specific viscosity η_sp/c of glutamate dehydrogenase continuously increases with increasing enzyme concentration, from about 4 ml/g at the lowest concentrations to about 16 ml/g at 8 mg/ml. In the presence of 10^?3M GTP and 10^?3M NADH the viscosity increase is much smaller and the results can be extrapolated to zero enzyme concentration to yield an intrinsic viscosity [η] = 3.2. The values of η_sp/c in phosphate buffer alone apparently extrapolate to the same value of [η], or to a value close to it. We also observe that, in the presence of toluene η_sp/c increases very much more with enzyme concentration: η_sp/c already equals 16 ml/g at a concentration of 0.75 mg/ml. These observations are in good agreement with the hypothesis that the active oligomer of glutamate dehydrogenase (MW = 312,000) associates with increasing enzyme concentration to form linear rodlike polymers of indefinite length. This association is strongly diminished by the addition of 10^?3M GTP, 10^?3M NADH. Toluene, on the other hand, promotes reversible association to linear polymers of very high molecular weight. The transverse and axial rotary frictional coefficients of macroscopic bodies, similar to a physical model for the structure of glutamate dehydrogenase recently advanced, were determined. Assuming that the viscosity of the model is equal to that of an ellipsoid of rotation with identical frictional coefficients, we calculate [η] = 3.26 ml/g according to Kuhn and 3.20 ml/g according to Simha, for the glutamate dehydrogenase oligomer, in good agreement with the result derived from the study of enzyme solutions.     

Viscous macromolecules inhibit erythrocyte hemolysis induced by snake venom phospholipase A2

The effect of medium viscosity on lysis of red blood cells (RBC) induced by snake venom phospholipase A2 (PLA2) was examined. The medium viscosity was modified by the addition of various macromolecules which differ in their chemical nature and in their capacity to increase fluid viscosity. PLA2 and Ca++ were applied to cells suspended in viscous medium to induce hemolysis. It was found that the hemolysis is inhibited in direct proportion to increasing viscosity of the extracellular fluid. This phenomenon was observed with aggregated as well as disaggregated RBC. To examine whether the viscosity interferes with the accessibility of the enzyme to the cell, the medium viscosity was modified after binding of the enzyme to the cells; PLA2 was added to a RBC suspension in the presence of Ba++ which binds the enzyme to the cell membrane but does not activate it. The cell-enzyme complex was separated by gel filtration and suspended in viscous medium in the presence of Ca++ which activates the reaction. Also in this case RBC lysis was inhibited as the medium viscosity was increased. It is proposed that the action of PLA2 on RBC membrane is regulated by the viscosity of the cell surface aqueous environment.     

Exogenous β-glucanases and pentosanases and their impact on mashing

Commercial enzymes differ in their content of β-glucanases and xylanases and, in their ability to reduce mash viscosity and increase extract. No simple relationship exists between measurable enzyme activity and these parameters, suggesting that the benefit of these enzymes in mashing is not simply predicted from absolute levels of enzyme, reinforcing the view that enzyme efficacy should be evaluated directly in mashes. Preparations comprising principally xylanase increase wort viscosity when added in lower levels to mashes, probably by releasing β-glucan from its entrapment by arabinoxylan within the cell walls of barley. The glucanases have greater impact than xylanases on viscosity. Combinations of xylanase and β-glucanase have the greatest impact on viscosity and extract yield.     

Modulation of egg-white lysozyme activity by viscosity intensifier additives

The activity of egg-white lysozyme was measured in the presence of carbohydrate additives in the reaction medium. These additives show a significant affinity for water. They depress water activity and increase the viscosity of the medium. Solute-solvent interactions in aqueous solutions of the additives are characterized by properties such as the intrinsic viscosity, Huggins constant apparent molar volume and hydration number. It was found that, despite the lowering of enzyme activity when the concentration of additive is increased, the behavior remains Michaelian and neither modification of Km nor inhibition by excess substrate is observed. On the other hand, the effect of the viscosity of the medium on enzyme activity was determined. This effect is independent of the nature of the additive at high viscosities (greater than 4 mPa s-1) for which enzyme activity is very low and appears to vary according to the kind of additive in dilute solution at low viscosities (less than 2 mPa s-1).     

The Role of Diffusion in Enzyme Kinetics

A discussion is given of the general role of diffusion in enzyme kinetics based upon a rigorous theory for bimolecular association and dissociation steps that was presented previously, and criteria are formulated for the dependence of the over-all rate on medium viscosity. With these criteria it is possible to conclude that a number of enzymes will exhibit no appreciable dependence of over-all rate on the medium viscosity, quite irrespective of the as yet unmeasured rate constants for association and dissociation of enzyme and substrate. The effect of adsorbing the enzyme onto the surface of a much larger spherical colloidal particle is considered with the conclusion that the rate will either remain the same or decrease, and that its sensitivity to medium viscosity will remain the same or increase.     

Rotational dynamics of the Ca-ATPase in sarcoplasmic reticulum studied by time-resolved phosphorescence anisotropy

We have investigated the microsecond rotational motions of the Ca-ATPase in rabbit skeletal sarcoplasmic reticulum (SR), by measuring the time-resolved phosphorescence anisotropy of erythrosin 5-isothiocyanate (ERITC) covalently and specifically attached to the enzyme. Over a wide range of solvent conditions and temperatures, the phosphorescence anisotropy decay was best fit by a sum of three exponentials plus a constant term. At 4 degrees C, the rotational correlation times were phi 1 = 13 +/- 3 microseconds, phi 2 = 77 +/- 11 microseconds, and phi 3 = 314 +/- 23 microseconds. Increasing the solution viscosity with glycerol caused very little effect on the correlation times, while decreasing the lipid viscosity with diethyl ether decreased the correlation times substantially, indicating that the decay corresponds to rotation of the protein within the membrane, not to vesicle tumbling. The normalized residual anisotropy (A infinity) is insensitive to viscosity and temperature changes, supporting the model of uniaxial rotation of the protein about the membrane normal. The value of A infinity (0.20 +/- .02) indicates that each of the three decay components can be analyzed as a separate rotational species, with the preexponential factor Ai equal to 1.25X the mole fraction. An empirically accurate measurement of the membrane lipid viscosity was obtained, permitting a theoretical analysis of the correlation times in terms of the sizes of the rotating species. At 4 degrees C, the dominant correlation time (phi 3) is too large for a Ca-ATPase monomer, strongly suggesting that the enzyme is primarily aggregated (oligomeric).(ABSTRACT TRUNCATED AT 250 WORDS)     

Modeling enzyme production with Aspergillus oryzae in pilot scale vessels with different agitation, aeration, and agitator types

The purpose of this article is to demonstrate how a model can be constructed such that the progress of a submerged fed-batch fermentation of a filamentous fungus can be predicted with acceptable accuracy. The studied process was enzyme production with Aspergillus oryzae in 550 L pilot plant stirred tank reactors. Different conditions of agitation and aeration were employed as well as two different impeller geometries. The limiting factor for the productivity was oxygen supply to the fermentation broth, and the carbon substrate feed flow rate was controlled by the dissolved oxygen tension. In order to predict the available oxygen transfer in the system, the stoichiometry of the reaction equation including maintenance substrate consumption was first determined. Mainly based on the biomass concentration a viscosity prediction model was constructed, because rising viscosity of the fermentation broth due to hyphal growth of the fungus leads to significant lower mass transfer towards the end of the fermentation process. Each compartment of the model was shown to predict the experimental results well. The overall model can be used to predict key process parameters at varying fermentation conditions.     

Medium viscosity regulates the activity of membrane-bound and soluble phospholipase A2

Medium viscosity is a regulator of very low density lipoprotein production by cultured hepatocytes; their secretion and synthesis are inversely proportional to the extracellular fluid viscosity. The possibility that the mechanism of this extracellular effect on cell function involves modulation of cell membrane component(s) was considered. Along with this assumption, we studied the effect of medium viscosity on the activity of phospholipase A2 (PLA2), an enzyme present in the cell surface membrane, and the activity has been correlated with cellular secretion. We have found that culture medium viscosity inhibits the activity of PLA2 in the plasma membrane of cultured liver cells, concomitantly with the inhibition of lysosomal enzyme and lipoprotein secretion. It was also found that the degradation of liposomal phosphatidylcholine by soluble snake venom PLA2 is inversely proportional to the solvent viscosity. The possibility that the effect of medium viscosity on the enzymatic reaction involves the modulation of dynamic properties of membrane phospholipids was then considered. This hypothesis was examined by monitoring the fluorescence depolarization of fluorophores incorporated into phospholipid vesicles. No significant effect of the solvent viscosity on the phospholipid bilayer was observed. It is proposed that the regulation of cellular secretion by extracellular fluid viscosity involves modulation of the cell membrane PLA2 activity.     

Diffusion-dependent rates for the hydrolysis reaction catalyzed by glyoxalase II from rat erythrocytes

Glyoxalase II from rat erythrocytes is a near optimal catalyst for the hydrolysis of S-D-lactoylglutathione in the sense that the magnitude of kcat/Km is limited, in large part, by the rate constant for diffusion-controlled encounter between substrate and active site. The experimental basis for this conclusion is derived from the dependencies of the kinetic properties of the enzyme on solution viscosity (pH 7, Ic = 0.1 M, 25 degrees C). When sucrose is used as a viscogenic agent, kcat/Km for S-D-lactoylglutathione (8.8 x 10(5) M-1 s-1) decreases markedly with increasing solution viscosity. This effect appears not to be due to a sucrose-induced change in the intrinsic kinetic properties of the enzyme, since kcat/Km for the slow substrate S-acetylglutathione (3.7 x 10(4) M-1 s-1) is nearly independent of solution viscosity. Quantitative treatment of the data using Stoke's law indicates that the rate of hydrolysis of S-D-lactoylglutathione will be approximately 50% diffusion limited when [substrate] much less than Km; the encounter complex between enzyme and substrate partitions nearly equally between product formation and dissociation to form free enzyme and substrate. The same conclusion is reached when glycerol is used as a viscogenic agent, once the apparent activation effect of glycerol on the intrinsic activity of the enzyme is taken into account. Finally, the rate of formation of the encounter complex between substrate and active site may be governed to a significant extent by charge-charge interactions.(ABSTRACT TRUNCATED AT 250 WORDS)     

The detection of hyaluronidase on electrophoresis membranes

A method is described for the detection of hyaluronidase enzyme activity following electrophoresis on cellulose acetate membranes prior to elution of the enzyme from the support medium. It appears to be specific for endo-N-acetylhexosaminidase activity. It detects 0.005 of the least amount of enzyme detected by a miniaturized turbidity reduction test or by a standard viscosity reduction test.     

Enzymatic hydrolysis of waste office paper using viscosity as operating parameter

Enzymatic hydrolysis of waste office (WO) paper with feeding WO paper in a reactor was investigated using apparent viscosity as operating parameter. Since the apparent viscosity was correlated with the concentration of pulping WO paper, the amount of hydrolyzed WO paper was assumed by measuring the decrease in the apparent viscosity. Then the amount of hydrolysis WO paper and the amount of enzyme corresponding to the desired ratio were fed into the reactor. When the WO paper and 1% (to the amount of WO paper) enzyme were fed to the hydrolytic reaction, 87 g/L of reducing sugar (RS) with a hydrolytic yield of 42.2% was obtained for a 24-h hydrolysis. However, when nonpulping WO paper and 5% (to the amount of WO paper) enzyme were fed to the hydrolytic reaction, 120 g/L of RS with a hydrolytic yield of 40% was obtained for a 24-h hydrolysis. Therefore, the RS concentration from this hydrolysis process feeding WO paper using apparent viscosity as operating parameter may be of sufficient concentration to serve as a carbon source in microorganism culture or chemical feedstock.     

杜仲绿原酸对高脂小鼠血液流变学作用研究

旨以研究杜仲绿原酸对高脂高胆固醇诱导的高血脂模型小鼠血液流变学的影响,以昆明小鼠为实验动物,随机分成5组:阴性对照组,模型对照组和低剂量(25 mg/kg BW)、中剂量(50 mg/kg BW)、高剂量(100 mg/kg BW)杜仲绿原酸组,每组10只.后4组饲以高脂饲粮,同时小鼠灌胃杜仲绿原酸4周,实验结束,分别测定各组小鼠血液流变学参数、血清和肝脏的抗氧化酶活性和脂质过氧化产物MDA含量及其总抗氧化能力和羟自由基清除率.高脂血症小鼠的全血粘度、血浆粘度、红细胞压积、血沉、纤维蛋白原、红细胞刚性指数和聚集指数显著降低(P＜0.05),红细胞变形指数显著提高(P＜0.05),小鼠血清和肝脏SOD、GSH-Px水平、总抗氧化能力和羟自由基清除能力均显著升高(P＜0.05),MDA水平显著降低(P＜0.05).在高脂膳食条件下,杜仲绿原酸能有效提高血液的抗氧化防御功能(包括抗氧化力、抗氧化酶活性)、改变血液流变学参数等,降低血液粘度、红细胞刚性和聚集,增强变形能力,使细胞膜的流动性增高,其中以中剂量效果相对较好.     

Viscosity and transient solvent accessibility of Trp-63 in the native conformation of lysozyme

We have measured the rates of isotope exchange at the nitrogen of the indole ring of Trp-63 of lysozyme and of L-tryptophan as a function of solution viscosity. We have used two cosolvents, glycerol and ethylene glycol, to modify the relative viscosity. We have derived the appropriate kinetic equations for the alternative possibilities that the exchange takes place either in solution or in the intact protein matrix. Because we chose to study the proton-catalyzed exchange reaction, the rate of it is not expected to be diffusion-limited. We confirmed this by measuring the exchange from tryptophan. These results and the known effects of glycerol and ethylene glycol on the solvation of indole allow us to predict that if the exchange reaction takes place in a protein matrix the effects of the two cosolvents when compared under isoviscous conditions should be identical. This is what we find for Trp-63 in lysozyme at 15, 20 and 26 degrees C. The slope of the linear plot of log k vs. log relative viscosity is 0.6. This strongly supports a model for conformational fluctuations where transient solvation takes place without major changes in protein folding. The most interesting feature of our findings is the fact that a slow reaction admittedly not diffusion-limited shows, when taking place in a protein matrix, a linear dependence on solution viscosity. We suggest that what we observe is the effect of damping of movement of the side chain expressed as a change in the friction along the reaction coordinate in the corresponding phase space. The presence of such effects stresses the validity and usefulness of Kramers model of rate processes for reactions taking place in a protein matrix. Such behavior is predicted by several of the recently proposed general mechanisms of enzyme catalysis.     

黄原胶寡糖生物活性的研究

利用黄原胶降解菌Cellulom onassp.XT11生产的黄原胶降解酶,对黄原胶进行生物降解,生产具有不同粘度/还原末端比的黄原胶寡糖,并研究了黄原胶寡糖在清除羟基自由基、植物防卫反应中激活因子活性和对植物病原菌抑制能力等方面的生物活性,结果表明黄原胶寡糖具有清除羟基自由基能力,并能激活植物防卫系统以抵御病原菌的侵染,同时对野油菜黄单孢菌也具有抑菌活性。     

The effect of solvent viscosity on the rate-determining step of fatty acid synthetase

The overall activity of an animal fatty acid synthetase at the saturation level of substrate concentration decreased when the solvent viscosity, eta, of the reaction mixture was increased with viscogens such as glycerol, sucrose, and polyethylene glycol. The activity of the enzyme changed roughly proportional to eta-P, where p = 1.0 for glycerol, p = 0.66 for sucrose, and p less than 0.6 for polyethylene glycol with different molecular sizes. The thioesterase activity, which catalyzes the final partial reaction in the multifunctional enzyme, was not affected by 5-fold increase of solvent viscosity with sucrose. These results suggested that the rate-determining step of the enzyme other than the thioesterase reaction involves a microscopic transport step, the rate of which is influenced by the solvent viscosity. The microscopic transport step may be related to the transfer of the reaction intermediate from one active site to another or to the motion of a larger part of the enzyme requisite for the catalytic reaction. In the solution containing glycerol, the rate-determining motion was primarily diffusion limited since the inverse of the initial rate was proportional to eta, i.e., p = 1. Since the substrate concentration was at a saturation level in this experiment, the viscosity-dependent step cannot be the encounter between the enzyme and substrates, but must be intramolecular in origin, most probably the reaction catalyzed by beta-ketoacyl synthetase. In solutions containing other viscogens, however, p was less than 1.0, indicating a significant involvement of chemical steps in the rate-determining step as well. Bovine serum albumin, when used as a proteinic viscogen, also decreased the initial rate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies on the Chitinolytic Enzymes of Black-koji Mold

It has been found that glycol chitin is a suitable substrate for the viscometric determination of chitinase activity, because the viscosity of its aqueous solution is not affected by the presence of added salt and the changes of pH, differing from chitosan acetate and carboxy-methyl chitin used by earlier workers. Using this substrate the viscometric activity is determined, basing on the observation that the time required to halve the viscosity of reaction mixture is inversely proportional to the amount of enzyme used.     

An extracellular agarase from a Cytophaga species

1. An extracellular enzyme has been isolated from cultures of the bacterium growing on agar or porphyran. 2. Partial purification of the enzyme has been achieved by precipitation with ammonium sulphate followed by gel filtration on DEAE-Sephadex A-50. 3. The optimum conditions for the enzyme acting on porphyran are pH7·2 and a temperature of 40–41°. 4. The enzyme has an endoaction, producing a rapid decrease in viscosity of agar or porphyran solutions with little development of reducing power. 5. From the action of the enzyme on various polysaccharides, it is concluded that the enzyme is specific for the agarose structure.