Kinetic parameters of immobilized

A Han s plot curve indicates how much the immobilization process can be affected as medicated from the kinetic parameters of immobilized enzyme. That the Km value has become five times higher than that of the free one is a reflection of powerful diffusion limitation of the substrate. 

Table II. Kinetic Parameters of Immobilized OPH Enzymes for Paraoxon...

Table III. Kinetic parameters of immobilized OPH enzymes for DFP and demeton-S hydrolysis. ...

The experiments described above indicate that technology is available to couple SPR with mass spectrometry. These methods should be useful for protein-protein interaction mapping. For example, immobilized proteins can be used as hooks for fishing binding partners from complex protein mixtures under native conditions. The coupling of techniques can lead not only to the rapid identification of interacting proteins but will also provide information on the kinetic parameters of the interaction. This approach should serve as an excellent complement to the use of in vivo techniques such as the yeast two-hybrid system. 

Vieira, L.G.T., Zaiat, M., and Foresti, E., Intrinsic kinetic parameters of substrate utilization by anaerobic sludge along the horizontal-flow anaerobic immobilized sludge (HAIS) reactor. Environ. Technol. 1997 vol. 18, no. 9, pp. 953-957. 

Mass transfer can alter the observed kinetic parameter of enzyme reactions. Hints of this are provided by non-linear Lineweaver-Burk plots (or other linearization methods), non-linear Arrhenius plots, or differing Ku values for native and immobilized enzymes. Different expressions have been developed for the description of apparent Michaelis constants under the influence of external mass transfer limitations by Homby (1968) [Eq. (5.69)], Kobayashi (1971), [Eq. (5.70)], and Schuler (1972) [Eq. (5.71)]. 

Table 3 Kinetic parameters of native and immobilized P-D-glucosidase at 20°C and 40°C [23] ...

The significance of Eq. (28) is that the thermometric data can be used for evaluation of the kinetic parameters Km and K , and, thus, for rapid determination of the kinetic properties of IMB preparations. This had been performed in the previous work by investigating the kinetic properties of immobilized invertase [27]. Typical kinetic data from this study are presented in Fig. 7. In this... 

The use of kinetic assay methods requires several assumptions regarding the kinetic parameters of the immobilized enzyme and the Influence of diffusion on the results. As has been discussed earlier, for many substrate-immobilized enzyme combina-... 

Magnetic particles coated with copolymer of acrylamide and acrylonitrile has been prepared. Those particles were used as a matrix for a covalent binding of peroxidase. The periodic-oxidized enzyme was bound to the matrix by immobihza-tion procedure at 4°C for 18 h. The immobihzed enzyme showed relative activity of 86%. The following results were obtained for pH and optimum temperature of the immobilized enzyme - 7.0°C and 30°C, respectively. The analysis of the kinetic parameters of the immobilized enzyme showed values of V - 0.0517 M x 10 s and - 2.3 x 10" M (Yotova et al. 2008). 

Table 4 shows a comparison between the kinetic parameters of soluble and immobilized peroxidase and on magnetic particles. 

Where R is the carrier radius, Defrthe effective diffusion coefficient of the substrate, E is the enzyme concentration in the carrier, and kcat and Km are the kinetic parameters of an enzyme. From a practical standpoint it is important to remember that there are no diffusional limitations as long as substrate concentration S exceeds Km. This condition normally exists at the beginning of many processes catalyzed by immobilized enzymes. At the end of the process, when a substrate is depleted and effective Km may increase because of the product inhibition, the whole reaction may be limited by diffusion. 

Table 5 Kinetic parameters of the wild type and mutants of isomalto-dextranase for the hydrolysis of the dextran immobilized on the 27-MHz QCM , and relative enzyme activ- ...

Table 2 Kinetics parameters of thermal deactivation, at 50 and 60 °C, of soluble CALB, CALB immobilized by adsorption on coconut fiber and Novozyme 435. ...

Based on the obtained results, immobilized enzyme prepared at pH 4 (CALB-4) and 5 (CALB-5) were selected for thermal stability studies at 60 °C. Table 2 shows the kinetic parameters of thermal deactivation at 60 °C estimated by fitting deactivation model, Eq. 2, to experimental data. It can be observed that the deactivation profiles of CALB-4 and CALB-5 were similar, as ki and 2 are almost the same. However, by comparing the half-lives of CALB-4 and CALB-5 to CALB-7A, it can be observed that the biocatalyst prepared at pH 7 is 3- and 3.8-fold more stable than the ones prepared at pH 4 and 5, respectively. Considering that CALB has an optimum pH between 7 and 8 [35], when the enzyme is adsorbed on coconut fiber at pH 7, a favorable molecule conformation is preserved, being, therefore, more thermal stable than CALB-4 and CALB-5. Therefore, lipase immobilized at pH 7 was selected for fiirther kinetic, operational, and thermal stability studies. 

Marangoni AG (2003) Enzyme kinetics a modem approach. Wiley Intersdence, Hoboken, 229 pp Mesentsev AV, Lamzin VS, Tishkov VI et al. (1997) Effect of pH on kinetic parameters of NAD -dependent formate dehydrogenase. Biochem J 321(2) 475 80 Messing RA (1975) Immobilized enzymes for industrial reactors. Academic Press, New York,... 

Table 12-8. Characteristics of MX immobilized on polymers with grafted functional layers and kinetic parameters of ethylene polymerization (343 K). 

Table 12-10. Kinetic parameters of cyclohexene oxidation in the presence of immobilized ...

Recently, a fusion protein between a cellulose binding domain (CBOeios)> isolated from Clostridium cellulovorans, and OPH was shown to be capable of immobilization onto various cellulose materials (24). The use of cellulose as an immobilization matrix is advantageous due to its low cost, wide spread availability and non-toxic nature. The kinetic parameters of OPH fused to die CBD domain were essentially identical to die soluble protein with a 3.8 fold increase in from 0.058 to 0.220 and a 10.4% decrease in Kd from 3170 to 2840. Additionally, the immobilized fusion protein offered superior stability over that of soluble OPH, retaining over 85% activity over a period of 45 days (24). 

One of the most important parameters of an immobilized-carrier complex is stability of its activity. Catalytic activity of the complex diminishes with time because of leakage, desorption, deactivation, and the like. The half-life of the complex is often used to describe the activity stabihty. Even though there may be frequent exceptions, hn-ear decay is often assumed in treating the kinetics of activity decay of an immobilized complex. 

Chen K-C, Wu J-Y, Yang W-B et al (2003) Evaluation of effective diffusion coefficient and intrinsic kinetic parameters on azo dye biodegradation using PVA-immobilized cell beads. Biotechnol Bioeng 83 821-832... 

FIGURE 1.17. Cyclic voltammetry of slow electron transfer involving immobilized reactants and obeying a Butler Volmer law. Normalized current-potential curves as a function of the kinetic parameter (the number on each curve is the value of log A ) for a. — 0.5. Insert irreversible dimensionless response (applies whatever the value of a). 

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