Spacer molecule enzyme immobilization

The immobilization of enzymes on solid supports is the oldest of new biotechnologies. As for most of the chemical applications, the uses in this field require a multi-step modification. As a first layer a simple silane, mostly APTS, is used. In order to minimize sterical hindrance in the ultimate application reaction, a spacer molecule is often introduced between the active surface group and the immobilizing species. This may either be introduced at once, using a long-chain functional silane, or in a second step, after silanization. 

Affinity chromatography combines the analytical and chemical capacities of chemically bonded stationary phases and immobilized enzymes. Technology and methodology of both techniques are joined in the development of affinity stationary phases. Since steric requirements are even more determining than in simple immobilized enzyme systems, spacer molecules have great importance in these modifications. Commonly used spacer arms are summarized in figure 8.3. 

The lower relative activities of the enzymes immobilized without spacer, compared to those of the enzymes immobilized with spacers, suggest that the enzymes undergo greater denaturation when immobilized without spacer than with spacers. This may be explained in terms of structural deformation of the immobilized enzyme molecules as illustrated in... 

Figure 4. The covalently immobilized enzymes without spacer must undergo remarkable deformation in the lower surface concentration region, whereas the enzyme molecules immobilized with spacer may be protected from the heavy structural deformation even in the lower surface concentration region owing to the spacer chain. The increase in the relative activity of the enzyme immobilized without spacer with the increasing surface concentration may be ascribed to a reduced interaction with the substrate, because of high enzyme concentration during the immobilization reaction. In addition, the spacer on the carrier surface probably reduces the steric interference with the substrate binding process, especially toward high molecular weight substrates.

Recently, several studies have immobilized bioactive species, such as enzymes, peptides and proteins, directly onto the polymer surface. The polymer siuface is fimctionalized with one of the above techniques to introduce the desired functional groups to subsequently react with the bioactive molecule. Thus, the functional group or the grafted polymer chains act as a spacer molecule between the material sm-face and bioactive compound. This helps in reducing the steric hindrance and shields the biomolecule from the hydrophobic surface preventing protein denaturation and enhancing bioactivity. The intermediary molecular chemistry can influence the bioactive compound behavior on the material surface. This approach has been used for PDMS used as biosensors [37]. 

In many instances, the enzyme shows reduced activity when in close proximity to the slide surface. To address this problem, a molecule, bearing amino groups, can be used as a spacer. Possible candidates are arginine, polylysine, 1,12-diaminododecane, and similar molecules or amino-terminated dendrimers such as those of the PAMAM family which afford a greater density of amino residues. The protocol presented here is effective for the immobilization of glucose oxidase, organophosphorus hydrolase, acetylcholinesterase, and butyrylcholinesterase. An alternate protocol was developed for the immobilization of carbonic anhydrase. 

In view of the high cost of NAD(P)+/ NAD(P)H cofactors, practical applications require their immobilization together with the enzymes. The covalent coupling of natural NAD(P)+ cofactors to an organic support results, however, in a substantial decrease of their efficiency. MobiKty of the cofactor is vital for its efficient interaction with enzymes, so serious attention has been paid to the synthesis of artificial analogs of the NAD(P)+ cofactors carrying functional groups separated from the bioactive site of the cofactor by spacers [277, 278]. The spacer is usually linked to N-G position of the NAD(P)+ molecule, and should provide some flexibility for the bioactive part... 

The immobilized enzyme should undergo strong deformation, especially in the lower surface concentration region without spacer (Figure 8A), whereas the immobilized GOD molecule widi spacer (Figure 8B) must be protected from the structure deformation even in the lower si ace concentration region owing to the spacer effect... 

The pH effect on the activity of the LPL immobilized onto PAM microsphere was studied for pNPL hydrolysis in PBS at 37 C over a wide pH region. The results are presented in Figure 9, where it is seen that the immobilized LPL has the same optimum at pH 7.0 as the free one, but the pH range where the immobilized enzyme has high activities is considerably widened, probably due to diffusional limitation of the immobilized enzyme molecule. Again, the directly immobilized LPL displays a greater stability against pH than the free and the immobilized enzymes with spacer. 

The weight of the immobilized enzymes remaining after the last batch was found to be practically the same as the original one for both cases, with and without spacer, suggesting no leakage of the immobilized enzyme during the repeated uses. This gives an evidence for the covalent fixation of the enzyme molecules onto the polymeric carrier surface. 

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