Compared to the two aforementioned methods, site-specific immobilization can offer both highly specific orientation and reliable stability of the immobilized enzyme (Fig. 17b). The active sites of the immobilized enzymes face away from the support surface and a consequent higher activity can be obtained (Butterfield et al. 2001). A typical site-specific process utilizes the biotin-avidin interaction to immobilize enzymes. Firstly, a single biotin moiety is incorporated onto the appropriate place (opposite the active site) of the enzymes. The biotin is recognized by avidin, which is immobilized onto the membrane surface, and the enzymes are immobilized to the membrane in a specific orientation (Fig. 21). As an advanced technique, site-specific immobilization provides a very promising way to realize simultaneously high stability and activity. Unfortunately, site-specific
Fig. 21. Site-specific immobilization of enzyme by the biotin-avidin interaction. ft-Gal ft-Galactosidase functionalized enzymes are not easy or readily produced and are usually very expensive. Thus, to our knowledge, no study has been carried out on PPMM with this technique. However, it is predictable that with the current rapid advances in biology this technique will receive more and more consideration in the near future.
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