Immobilization parameters

Among the two hydrophobic supports, octadecyl-sepabeads presented the best result concerning YLL immobilized activity (15.5 U/g), as its activity was over twice higher than YLL immobilized onto octyl-agarose (7.0 U/g). The other immobilization parameters calculated (immobilization yield and activity retention) were also better for octadecyl sepabeads (Table 1). These results were probably related with the chemical nature of hydrophobic supports. When a very hydrophobic support like octadecyl-sepabeads is used, there might be an increment of the affinity between the support and the substrate that... 

Immobilization parameters (recovered activity and immobilization yield) were calculated for the biocatalyst with higher hydrolytic activities (adsorptions performed between pH 3 and 6), and results are presented in Fig. 4. It ean be observed that best results for recovered activity and immobilization yield were obtained at pH 4 and at 5 or 6, respectively. 

These results showed the benefits of systematic investigations of immobilization parameters to achieve enhanced enzyme-catalyst activities. However, much work remains to understand on a molecular level how, for example, changes in enzyme loading influences the fraction of active lipase. Details of enzyme conformation and orientation as function of surface chemistry and morphology on model systems taken together with better control of surface... 

Meter F, Zarcula C, Kiss C et al. (2007) Enhancement of lipases enantioselectivity by entrapment in hydrophobic sol-gel materials influence of sdane precursors and immobilization parameters. J Biotechnol 131(2) S109... 

In order to achieve an efficient biocatalytic composite, the immobilization parameters e.g., lipase concentration, pH of the immobilization phase, activation reagent) should be correlated with the support morphology and type of the functional groups on the support surface. In this way, the composite biocatalyst may exhibit a higher catalytic efficiency. For the... 

The advantage of this factorial design technique is the ability to examine the influence of aU immobilization parameters independently as they are orthogonal. Figure 14 indicates that half of the maximum binding capacity is achieved after 30 min, which means that the Tresyl activation is a fast reaction. 

Active immobilized enzyme investigations of the immobilization parameters and the kinetics and stability of the immobilized enzyme... 

Table 2. Typical Operating Parameters for Immobilized Glucose Isomerase and Penicillin V Acylase...

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. 

Table 12. Parameters of reversible oxygen transport for haemoglobin and a microdisperse form of immobilized haemoglobin (pH 7.4, 37 °C, pC02 = 40 Torr, p02 = 0-150 Torr)...

The immobilization procedure may alter the behavior of the enzyme (compared to its behavior in homogeneous solution). For example, the apparent parameters of an enzyme-catalyzed reaction (optimum temperature or pH, maximum velocity, etc.) may all be changed when an enzyme is immobilized. Improved stability may also accrue from the minimization of enzyme unfolding associated with the immobilization step. Overall, careful engineering of the enzyme microenvironment (on the surface) can be used to greatly enhance the sensor performance. More information on enzyme immobilization schemes can be found in several reviews (7,8). 

As a general rule, the optimal immobilization method is found empirically by a process of trial and error, where the selectivity, activity, and operational stability of the enzyme after immobilization are taken into account. The immobilization process is very sensitive to different parameters and is treated as a kind of art [16]. 

A specific example where heterogeneous supports provide nanoparticle size-control is the immobilization of homogeneous silver nanoparticles on polystyrene [366]. This work was extended later to the development of a one-pot method for the size-selective precipitation of silver nanoparticles on PVP-protected thiol-functionalized silica. During the immobilization of very small silver nanoclusters both the size of the silver nanoclusters and the thickness of the silver layer on the support could be controlled directly by the reaction parameters applied (Fi re 16) [367]. 

The ability of free or immobilized lipoxygenase to introduce oxygen derived from the air into polyunsaturated fatty acids in media containing organic solvent and aqueous buffer has been investigated [9,56]. The influence of many parameters was tested upon the degree of oxygenation in biphasic systems [36,108]. 

A very promising method, immobilized artificial membrane (IAM) chromatography, was developed by Pidgeon and co-workers [299-304,307], where silica resin was modified by covalent attachment of phospholipid-like groups to the surface. The retention parameters mimic the partitioning of drugs into phospholipid bilayers. The topic has been widely reviewed [47,298,307,309-311]. 

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. 

When the network junctions are entirely immobilized by the surrounding chains, h equals zero. Then the junctions in a deformed specimen are displaced in proportion to the macroscopic strain, i.e., the deformation is affine. Alternatively, h equals unity when junction fluctuations are not impeded, the defining characteristic of a phantom network (16, 17). The parameter h was introduced (13) to allow empirically for different degrees of fluctuations. For undiluted networks at small deformations, h should usually be small, though not necessarily zero. 

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