Protein immobilization yield

Rp protein immobilization yield (mg immobilized enzyme/mg contacted protein) Rp enzyme immobilization yield (units of enzyme activity expressed in the biocattilyst/unit of enzyme activity contacted) SA specific activity of the biocatalyst (units of enzyme activity expressed/unit mass of biocatalyst)... 

Hemoproteins are a broad class of redox-proteins that act as cofactors, e.g. cytochrome c, or as biocatalysts, e.g. peroxidases. Direct ET between peroxidases such as horseradish peroxidase, lactoperoxidase," or chloropcroxidasc"" and electrode surfaces, mainly carbonaceous materials, were extensively studied. The mechanistic aspects related with the immobilized peroxidases on electrode surfaces and their utilization in developing biosensor devices were reviewed in detail. The direct electrical contact of peroxidases with electrodes was attributed to the location of the heme site at the exterior of the protein that yields close contact with the electrode surface even though the biocatalyst is randomly deposited on the electrode. For example, it was reported " that non-oriented randomly deposited horseradish peroxidase on a graphite electrode resulted in 40-50% of the adsorbed biocatalyst in an electrically contacted configuration. For other hemoproteins such as cytochrome c it was found that the surface modification of the electrodes with promoter units such as pyridine units induced the binding of the hemoproteins in an orientation that facilitated direct electron transfer. By this method, the promoter sites induce a binding-ET process-desorption mechanism at the modified electrode. Alternatively, the site-specific covalent attachment of hemoproteins such as cytochrome c resulted in the orientation of the protein on the electrode surfaces and direct ET communication. ... 

As shown in Equation 1, the capacity per cycle is directly proportional to the amount of antibody immobilized, the immobilization yield, the M.W. of the protein and the column volume and an exponential function of the number of cycles. The amount of antibody immobilized will usually be less than 10 gL l. Higher activation of the matrix required for greater than 10 gL l loading results in a decrease in the immobilization yield. The maximum immobilization yield is 1.0 (100%) while 0.8 (80%) is not difficult to obtain. The M.W. of the protein to be isolated is fixed. The only way to increase the capacity per cycle significantly is to increase the volume of the immunosorbent or increase the number of cycles prior to reaching 50% of initial capacity (cycle half-life). Increasing the volume of immunosorbent increases the amount of monoclonal antibody required. 

Recovered activity and immobilization yield were calculated, and results are listed in Table 1. It can be observed that recovered activity increased when the initial enzyme concentration in the supernatant (Eq) was increased to 60 and 90 U/ml, but it remained almost constant for the other concentrations studied. Immobilization yield, on the other hand, decreased when high concentrations of enzyme were used (60 to 150 U/ml, compared to 30-40 U/ml). When Ef,=9Q U/ml, the highest value of recovered activity was obtained, which suggests that protein molecules are probably immobilized at close proximity to each other, which may prevent deactivation caused by enzyme unfolding by covering the support surface. In other words, when enzyme load was increased, more enzyme molecules were immobilized and less area of the support is available for lipase to spread itself, which may prevent loss in activity [31]. 

Nevertheless, when higher concentrations of lipase in the supernatant were used, Eo= 150 U/ml, recovered activity decreased, and immobilization yield was enhanced (Table 1). According to the literature [4, 6, 9, 10], protein adsorption is not restricted to a monolayer on the support, and adsorption of secondary layers has been reported. Therefore, when Eo= 150 U/ml, probably a second layer of lipase was adsorbed on the first layer, leading to an improvement on immobilization yield, as more enzyme molecules were adsorbed. However, although more molecules were immobilized on coconut fiber, not all of them... 

Although the amount of C. antarctica lipase type B adsorbed to coconut fiber was nearly independent on the pH of adsorption (between pH 3 and 6), immobilization yield and recovered activity were dependent on the pH of adsorption because interactions between the molecule and its environment influence the structure of a protein molecule, and these interactions are pH-dependent [8],... 

This method enables the rapid identification of DNA-binding proteins. Immobilized DNA probes harboring a specific sequence motif are incubated with cell or nuclear extract. Proteins are analyzed directly off the solid support by MALDl-TOP. The determined molecular masses are often sufficient for identification. If not, the proteins are subject to MS peptide mapping followed by database searches. Apart from protein identification, the protocol also yields information on post-translational modifications. The protocol was validated by the identification of known prokaryotic and eukaryotic DNA-binding proteins, and is use provided evidence that poly(ADP-ribose) polymerase exhibits DNA sequence-specific binding to DNA. ... 

During the process of immobilization a fraction of the enzyme protein is immobilized, while the rest remains unbound. The immobilized enzyme protein expresses only a fraction of the expected activity and this can be due to enzyme inactivation, steric hindrance or mass transfer limitations. On the other hand the unbound enzyme protein may be partly inactive, so enzyme immobilization yield (Ye), as defined by Eq. 4.1, has to be carefully analyzed in its meaning. 

Both hydrophobic supports were challenged at higher protein loads. As seen in Table 6.3.3, specific activity of the biocatalyst could be substantially increased, but no interfacial activation was revealed at higher loads. Butyl Sepabeads at 1.75 mg protein/g support was considered the best biocatalyst for having the highest specific activity at a still high enzyme immobilization yield besides, the biocatalyst is easily... 

Gel Chlorella Sp %Protein immobilized 1st Cycle 2nd Cycle (Yield %)... 

The ELP expression system was compared to the conventional oligohistidme fusion, which is traditionally applied for purification by immobilized metal affinity chromatography (IMAC). Both techniques were shown to have a similar yield of the recombinant protein. The temperature-triggered approach offers a fast and inexpensive nonchromatographic separation with the possibility for larger scale purification. Although the ELP expression system may not be applicable to all types of recombinant proteins, numerous examples have already been shown [40]. 

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