Cross-linking of enzymes

Like many other useful discoveries, enzyme immobilization by cross-linking was actually an unintended by-product of another research project. In 1964, Florante Quiocho and Frederic Richards at Yale university cross-linked crystals of carboxy-peptidase-A with glutaraldehyde (pentane-1,5-dial), hoping to get stable crystals for X-ray diffraction studies. They noted that these cross-linked enzyme crystals (now 

For most enzymes, the CLEC is much more robust than the simple isolated enzyme. CLECs can withstand higher temperatures, they denature more slowly in organic solvents, and they are less susceptible to proteolysis [71]. Moreover, since there is no external support involved, CLECs exhibit a high volumetric productivity. These advantages, together with the tunable particle size (typically 1-100 pm), make CLECs attractive for industrial biocatalysis applications. 

---

Figure 6. Cross-linking of enzymes with glutaraldehyde.

Effective and simple immobilization of enzymes can be obtained by the cross-linking of enzyme aggregates, so-called CLEAs [55]. In this way, essentially any enzyme, including crude preparations, can be transformed into a heterogeneous type of material, insoluble in both water and organic solvents, that is stable and recyclable with high retention of the enzyme s original activity [56], These enzyme preparations are, therefore, of special value for both bio-bio and bio-chemo cascade processes. 

In order to give a homogeneous distribution of enzyme molecules inside the membrane, it was necessary to synthesize the membrane and to incorporate the enzymes at the same time. The co-cross-linking of enzyme molecules with an inert protein appears to be a proper solution. Purely active proteic films were created by using this procedure.11-12 These artificial enzyme membranes can be used in the study of heterogeneous enzyme kinetics and for modeling biological membranes. The phenomena in the enzyme membranes can be classified in two parts. 

Fig. 2.19 Glutaraldehyde cross-linking of enzyme E with bovine serum albumin (BSA), followed by Li borohydride stabilization...

Chemical methods that involve the formation of at least one covalent bond (attachment of enzyme to water-insoluble functionalized polymers, intermolecular cross-linking of enzyme molecules using multifunctional... 

An extreme case of covalent binding is cross-linking of enzymes. Instead of fixing the enzyme to a carrier, the enzyme acts as a carrier itself Enzyme aggregates or crystals, enzymes in a spray-dried form, or even enzymes in solution can be cross-linked. The immobilized enzyme is carrier free, that is the material is virtually pure enzyme and the negative effects of carriers can thus be avoided [10, 70]. 

Man-made usage of binding enzymes onto solid materials goes back to the 1950s, when immobilized enzymes, that is enzymes with restricted mobility, were first prepared intentionally [1,2]. Immobilization was achieved by inclusion into polymeric matrices or binding onto carrier materials. Considerable effort was also put into the cross-linking of enzymes, either by cross-linking of protein alone or with the addition of inert materials [3]. 

In the course of the last decades numerous methods of immobihzation on a variety of different materials have been developed. Binding to pre-fabricated carrier materials appears to have been the preferred method so far. Recently, cross-linking of enzyme crystals has also been reported to be an interesting alternative [4]. 

Cross-linking of enzyme molecules (e.g., with glutaraldehyde)... 

Immobilization by cross-linking of enzyme crystals appears to be a generic method however, unique protocols must be developed for each individual protein. Preparation of a CLEC form of many types of proteins and classes of enzymes have been reported including hydrolases, oxidoreductases, carbon-carbon lyases and isomerases. Crystallization of the protein is a highly effective purification step, so... 

Immobilization of enzymes by cross-linking with bifunctional reagents is carried out by different methods direct cross-linking of enzymes cross-linking of enzymes with inert proteins adsorption of enzymes on a water-insoluble carrier with subsequent processing using a bifunctional reagent. Albumin is... 

Govardhan, C.P., 1999. Cross-linking of enzymes for improved stability and performance. Curr. Opin. Biotechnol. 10, 331—335. 

---