Enzyme ionic strength

Environment temperature, light, presence of enzymes, ionic strength, and hydrogen ion concentration... 

Many enzymes need a certain ionic strength to maintain an optimum stabiHty and solubiHty, eg, bacterial a-amylases show optimal stabiHty in the presence of 1—2% NaCl. Some enzymes may need certain cations in low amounts for stabilization, eg, Ca " is known to stabilize subtiHsins and many bacterial a-amylases. Antioxidants (qv) such as sodium sulfite can stabilize cysteine-containing enzymes which, like papain, are often easily oxidized. 

Physica.1 Sta.bihty, Physical stabiHty depends primarily on the purity of the enzyme. Impurities remaining from the fermentation broth may precipitate or form a hazy solution. Unwanted sedimentation is often related to Ca " or acidic polysaccharides. The solubiHty of some enzymes can be increased by optimizing the ionic strength or changing the dielectric constant of the solution by a dding low molecular-weight polyols. 

Most ingredients in a detergent formulation contribute to the ionic strength of the wash solution. The effect of ionic strength on protease performance depends on pH and enzyme identity. The pH wash solutions also affects protease performance (Pig. 8). 

Fig. 8. Protease washing performance in a U.S. liquid detergent. Grass soiling in a 10 min wash at 30°C with one enzyme dosage, (a) pH profile of commercial proteases A and B. (b) Effect of increasing ionic strength, adjusted with Na2S04, of commercial protease B at (—°—) pH 8 and (- pH 11.

FIGURE 14.7 Substrate saturation curve for au euzyme-catalyzed reaction. The amount of enzyme is constant, and the velocity of the reaction is determined at various substrate concentrations. The reaction rate, v, as a function of [S] is described by a rectangular hyperbola. At very high [S], v= Fnax- That is, the velocity is limited only by conditions (temperature, pH, ionic strength) and by the amount of enzyme present becomes independent of [S]. Such a condition is termed zero-order kinetics. Under zero-order conditions, velocity is directly dependent on [enzyme]. The H9O molecule provides a rough guide to scale. The substrate is bound at the active site of the enzyme. 

In many situations, the actual molar amount of the enzyme is not known. However, its amount can be expressed in terms of the activity observed. The International Commission on Enzymes defines One International Unit of enzyme as the amount that catalyzes the formation of one micromole of product in one minute. (Because enzymes are very sensitive to factors such as pH, temperature, and ionic strength, the conditions of assay must be specified.) Another definition for units of enzyme activity is the katal. One katal is that amount of enzyme catalyzing the conversion of one mole of substrate to product in one second. Thus, one katal equals 6X10 international units. 

Structured laundry liquids are currently available in Europe and were recently introduced in the United States [50,51]. These products typically contain high levels of surfactants and builder salts, as well as enzymes and other additives. In the presence of high ionic strength, the combination of certain anionic and nonionic surfactants form lamellar liquid crystals. Under the microscope (electron microscope, freeze fracturing) these appear as round droplets with an onion-like, multilayered structure. Formation of these droplets or sperulites permits the incorporation of high levels of surfactants and builders in a pourable liquid form. Stability of the dispersion is enhanced by the addition of polymers that absorb onto the droplet surface to reduce aggregation. 

Enzymatic reactions are influenced by a variety of solution conditions that must be well controlled in HTS assays. Buffer components, pH, ionic strength, solvent polarity, viscosity, and temperature can all influence the initial velocity and the interactions of enzymes with substrate and inhibitor molecules. Space does not permit a comprehensive discussion of these factors, but a more detailed presentation can be found in the text by Copeland (2000). Here we simply make the recommendation that all of these solution conditions be optimized in the course of assay development. It is worth noting that there can be differences in optimal conditions for enzyme stability and enzyme activity. For example, the initial velocity may be greatest at 37°C and pH 5.0, but one may find that the enzyme denatures during the course of the assay time under these conditions. In situations like this one must experimentally determine the best compromise between reaction rate and protein stability. Again, a more detailed discussion of this issue, and methods for diagnosing enzyme denaturation during reaction can be found in Copeland (2000). 

The enzymes differ significantly in their optimal conditions for enzymatic activity. These include pH, ionic strength and temperature. The variety of conditions enabled us to verify if there could be limits to the approach. 

Since Diaz and Balkus first attempted to immobilize enzymes on mesoporous MCM-41 [101], several research groups have investigated the influence of various physical factors such as pore size, ambient pH, and ionic strength, on the adsorption efficiency of proteins [102-118]. This research revealed the general tendencies of protein adsorption behavior and outlines for successful immobilization of proteins onto mesoporous materials. As one of the representative examples, systematic... 

The kinetics of the general enzyme-catalyzed reaction (equation 10.1-1) may be simple or complex, depending upon the enzyme and substrate concentrations, the presence/absence of inhibitors and/or cofactors, and upon temperature, shear, ionic strength, and pH. The simplest form of the rate law for enzyme reactions was proposed by Henri (1902), and a mechanism was proposed by Michaelis and Menten (1913), which was later extended by Briggs and Haldane (1925). The mechanism is usually referred to as the Michaelis-Menten mechanism or model. It is a two-step mechanism, the first step being a rapid, reversible formation of an enzyme-substrate complex, ES, followed by a slow, rate-determining decomposition step to form the product and reproduce the enzyme ... 

FIGURE 5.7. Effect of changing the cosubstrate and the pH on the kinetics of an homogeneous redox enzyme reaction as exemplified by the electrochemical oxidation of glucose by glucose oxidase mediated by one-electron redox cosubstrates, ferricinium methanol ( ), + ferricinium carboxylate ( ), and (dimethylammonio)ferricinium ( ). Variation of the rate constant, k3, with pH. Ionic strength, 0.1 M temperature 25°C. Adapted from Figure 3 in reference 11, with permission from the American Chemical Society. 

---