Enzyme activity reaction conductance

An alternative containment scheme is immobilization of active species on a surface" " or within a tethered polymer brush or network. ° Surface immobilization can achieve high surface utilization by locating mediators and biocatalysts within nanometers of conducting surfaces. Immobilization on polymer networks allows for dense packing of enzymes within electrode volumes at the expense of long-distance electron mediation between the enzyme active center and a conductive surface. Such mediation often represents the rate-limiting step in the overall electrode reaction. 

The asymmetric hydrolysis of (exo,exo)-7-oxabicyclo[2.2.1]heptane-2,3-dimethanol, diacetate ester (37) to the corresponding chiral monoacetate ester (38) (Fig. 12B) has been demonstrated with lipases [61]. Lipase PS-30 from P. cepacia was most effective in asymmetric hydrolysis to obtain the desired enantiomer of monoacetate ester. The reaction yield of 75 M% and e.e. of >99% were obtained when the reaction was conducted in a biphasic system with 10% toluene at 5 g/liter of the substrate. Lipase PS-30 was immobilized on Accurel PP and the immobilized enzyme was reused (5 cycles) without loss of enzyme activity, productivity, or e.e. of product (38). The reaction process was scaled up to 80 liters (400 g of substrate) and monoacetate ester (38) was isolated in 80 M% yield with 99.3% e.e. The product was isolated in 99.5% chemical purity. The chiral monoacetate ester (38) was oxidized to its corresponding aldehyde and subsequently hydrolyzed to give chiral lactol (33) (Fig. 12B). The chiral lactol (33) obtained by this enzymatic process was used in chemoenzymatic synthesis of thromboxane A2 antagonist (35). 

To explore whether the presence of glucose in the reaction mixture of the AO activity measure could negatively affect the enzymatic activity of AO by means of an inhibitory effect, we conducted several experiments in which we added different amounts of glucose to the reaction mixture in order to determine enzyme activity. The results, shown in Table 4, clearly demonstrate that the presence of glucose at any concentration had no effect on AO activity. 

A series of enzyme-catalyzed reactions recently conducted in both conventional and supercritical fluid medium has shown that while no loss of enzyme activity was experimentally observed for the conventional medium, the same was no longer valid for supercritical C02 systems (1,4,10,11). For instance, Steinberger and Marr (12) have pointed out that the stability of an enzyme in supercritical C02 depends onboth its tertiary structure and several parameters during exposure to high-pressure fluid. They argued that high temperatures, the water content in C02 and pressurization/de-pressurization steps might cause enzyme inactivation. 

Computational approach. Lee and Houk conducted calculations using a methyl-ammonium ion to mimic the key lysine of the enzyme active site.16 They chose this model because, even though no crystal structures had been solved at the time, a lysine was known to be essential for catalysis.60 The reaction of orotate + CH3NH3 to form a carbene-methylamine complex was thus examined in various dielectrics using the SCI-PCM SCRF method in Gaussian 94.30 31 48 Solvation energies computed at the RHF/6-31 + G level were used to correct gas phase MP2/ 6-31 + G energies and obtain AH values for reaction in solution. 

With this configuration the conducting polymer serves as an inunobilisation platform for the enzyme. The potential applied to the Reaction Zone side of the membrane can be independently optimised to ensure maximum enzyme activity. 

Because the costs of isolation and purification of soluble enzymes are high and it is often both technically difficult and costly to recover an active form of the enzyme from product mixtures when the reaction of interest is completed, soluble enzymes are normally employed only in batch operations in which the enzymes are removed from the liquid product by precipitation. Thermal deactivation may be used instead to destroy the catalytic activity of the enzyme. Immobilization of the enzyme circumvents these difficulties because the solid phase containing the enzyme is easily recovered from the product mixture. Use of immobilized enzymes makes it possible to conduct the process in a continuous flow mode, thereby facilitating process control via manipulation of the flow rate of the process stream. One can offset losses in enzyme activity as time elapses by reducing the flow rate to maintain a constant product composition. Operation in this mode permits one to obtain more product per unit of enzyme employed. 

When reaction (22) is 50—100 times faster than reaction (21), the oxygen consumption is proportional to the activity of L-alanine dehydrogenase (Figure 8). Since NAD+ is continuously regenerated, the enzyme activity is proportional to time, and does not show the typical progressive inhibition due to the accumulation of reduced pyridine coenzyme. This technique allows many dehydrogenases be studied under simple experimental conditions. Moreover, in the presence of excess pyridine-linked dehydrogenase, experiments can be conducted with very low substrate concentrations (1—5 nmol). 

CGTase activity was measured at the following temperatures (40, 50, 70, 80, and 90 °C) and pH 6.0 (pH of maximum catalytic activity), keeping fixed the biocatalyst mass (307.5 mg). Samples of the reaction medium were taken in duplicates at the times of 0 and 25 min and added to a test tube containing 20 pL of 5 M HCl. These tubes were immersed in boiling water for 5 min to inactivate the enzyme. After cooling, a sample was taken to determine P-CD concentration using the phenolphthalein colorimetric method. The same assay was conducted from pH 4.0 to 10.0 and 60 °C, the temperature for maximum enzyme activity. 

The activity of the native and modified SC was also studied for the reaction methanolysis of APEE, using the substrate, methanol, itself as a solvent. This was done for three different salt-enzyme preparations, consisting of 99% salt, 50% salt, and no added salt (except that present in buffer). It was found that the PEG modification resulted in a six- to sevenfold increase in the initial rates of conversion for the cases with no surfactant and with T 20 (Table 2). AOT was found to reduce the activity of the enzyme preparations in all experiments conducted in the solvent methanol. Previous reports on enzymatic conversion in organic solvents have shown the effect of the solvent dielectric constant on enzyme activity for salt-free enzymes [22], Relationship between activity of AOT and PEG-modified SC to the hydrophobicity coefficient of various solvents has also been studied [20], however, only for the enzymes without salt-lyophilization. The decrease in activity of enzymes in organic solvents is attributed to the decreased water availability in organic media. Additionally, as the dielectric constant increases, the potential for removal of the... 

FIGURE 5 Influence of tetrahydrobiopterin (H4B) on the inhibitory action of nitric oxide (NO) on nNOS activity. Standard enzyme incubations were conducted for 10 min as described previously (Griscavage et al., 1994) and are similar to the conditions in Fig. 1. NO was added to reaction mixtures immediately after addition of NOS. H4B was added to reaction mixtures just prior to initiation of reactions by addition of NOS. Control reaction mixtures contained 10 fiM H4B. Data represent the means se of duplicate determinations from four separate experiments. 

Inspection of Table 2, entries 1-4, shows that optimum conversion and polyester molecular weight from 1 were at 70 °C. HiC activity dropped precipitously for polymerizations conducted at 80 °C. These results agree with those described above for condensation polymerizations. By performing poly-(e-caprolactone) in toluene instead of in bulk at 70 "C, Mi increased from 16 000 to 24 900 and Afw/Mi decreased from 3.1 to 1.7. An increase in Mi is expected for solution polymerizations since the solvent decreases the viscosity of the reaction medium, thereby easing diffusion constraints between substrates and the enzyme. However, the decrease in polydispersity for the solution polymerizations is less easily explained. One possibility is that transesterification reactions leading to broader polydispersity occur more rapidly for reactions conducted in bulk. HiC catalysis of a>-pentadecalactone (2) polymerization in... 

A complex and radically new situation evolves in the case of a direct, mediatorless, transport between the enzyme active center and the electrode. Apart from the problems mentioned above, some new fundamental questions arise, which have not been encountered either in electrochemistry or enzy-mology. In the case of preservation of the molecular integrity of the immobilized enzyme, electrochemical transformations of the substrate in this system take place at large (some 10-A) distances from the conductive phase. Therefore, it is necessary to investigate the mechanism of electron transfer and of the distribution of the potential jump (the structure of the electric double layer) in the electrode-enzyme-electrolyte system. The electrode becomes the donor or acceptor of electrons when the reaction proceeds at the enzyme active center. This implies a change in the functioning mechanism of the enzyme as compared to the native conditions. The chemical and electronic structure of the electrode surface must play an extremely important role in... 

---