Enzymes stabilizing factors

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). 

It appears that qualitative correlations between antibacterial activity and rate constants of HO ion catalyzed hydrolysis are fortuitous since many factors other than transpeptidase acylation contribute to antimicrobial activity. These other contributing factors include permeation of the outer membrane of the bacterial cell wall, resistance to /3-lactamase, the fit in the active site of the enzyme, stability of the acylated enzyme, and, last but not least, in vivo pharmacokinetic behavior. 

This volume is then brought to the specified 1 mL final volume with the addition of 0.5 mL deionized water (or buffer and any other factors needed to maintain enzyme stability). 

In vitro enzymatic polymerizations have the potential for processes that are more regio-selective and stereoselective, proceed under more moderate conditions, and are more benign toward the environment than the traditional chemical processes. However, little of this potential has been realized. A major problem is that the reaction rates are slow compared to non-enzymatic processes. Enzymatic polymerizations are limited to moderate temperatures (often no higher than 50-75°C) because enzymes are denaturated and deactivated at higher temperatures. Also, the effective concentrations of enzymes in many systems are low because the enzymes are not soluble. Research efforts to address these factors include enzyme immobilization to increase enzyme stability and activity, solubilization of enzymes by association with a surfactant or covalent bonding with an appropriate compound, and genetic engineering of enzymes to tailor their catalytic activity to specific applications. 

The liver (Cu,Zn)-SOD of old rats (27 months) showed a 60% reduction of specific activity, the presence of antigenically cross-reacting material, and a decreased thermal stability in comparison with the enzyme of young rats (6 months) . Similar observations were made with ageing human fibroblasts in culture, whereby the absence of a cytosolic stabilizing factor was suspected in old cells... 

There are many factors that influence the outcome of enzymatic reactions in carbon dioxide. These include enzyme activity, enzyme stability, temperature, pH, pressure, diffusional limitations of a two-phase heterogeneous mixture, solubility of enzyme and/or substrates, water content of the reaction system, and flow rate of carbon dioxide (continuous and semibatch reactions). It is important to understand the aspects that control and limit biocatalysis in carbon dioxide if one wants to improve upon the process. This chapter serves as a brief introduction to enzyme chemistry in carbon dioxide. The advantages and disadvantages of running reactions in this medium, as well as the factors that influence reactions, are all presented. Many of the reactions studied in this area are summarized in a manner that is easy to read and referenced in Table 6.1. 

Alcohol oxidase. We have chosen to use alcohol oxidase from Hansenula polv-morpha (2) in our research. The enzyme has eight sub-units, all of which must be associated for the enzyme to exhibit activity. It is a large enzyme Mn 600,000 and has poor stability upon freeze drying. This factor combined with an almost flat activity response to pH between 6 and 10 and a maximum temperature activity at 40-50°C made the enzyme an ideal candidate for the study of enzyme stabilization in the dry state, especially as we wished to prepare diagnostic kits for ethanol and solid phase enzyme based sensors. 

Different industries pose different challenges to the protein formulator. Many feed enzymes are sold as formulated liquid concentrates. In this case, the major requirements for a liquid formulation are enzymatic stability and preservation against microbial growth. It is sometimes not appreciated that the dominant factor affecting enzyme stability is the intrinsic stability of the enzyme itself formulation can do very little to correct for a structurally labile protein. Therefore, it is advisable to make stability an important criterion of the initial enzyme screening process. 

Especially in case of hydrophobic monomers, solubility becomes a limiting factor for the efficiency of enzyme-initiated polymerizations. Biocatalyst solubilization in organic media may be a viable solution as proposed recently by Li and coworkers [29]. Polar organic solvents can be used to some extend to increase the monomer solubility in the reaction medium [12,30]. However, the delicate balance between enhanced monomer solubility on the one hand and decreased enzyme stability in the presence of water soluble organic solvents on the other hand [27] has to be considered. Emulsion polymerizations have been reported for highly unpolar substrates such as styrenes [14, 21, 31] or use of unconventional reaction media such as supercritical C02 [19]. 

Most carboxylations and decarboxylations involve anionic intermediates [cf. Eq. (2)]. The stability of the anion is an important factor in determining reaction rates. An unstable anion will be very reactive toward CO2, but it will be difficult to form. A stable anion will be less reactive toward CO2, but it will be easy to form. Many carboxylations involve removal of a proton from an acid of pKa 15-18. Enzymic stabilization of such anions is very important. 

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