Carrier-Free Immobilized Enzymes

Enzyme biocomposites can also be designed without the participation of any solid support [108,109]. Such carrier-free immobilized enzymes can be prepared by direct cross-linking of the protein molecules in different physical states (e.g., dissolved, crystalline, spray-dried, or physically aggregated enzymes) leading to various structures [110]. 

Christakopoulos and Sheldon groups reported feruloyl and acetylxylan esterases enzymes immobilized following the CLEA biocomposites concept. 

In conclusion, the differences between the CLEMPA and lEMP concepts [99] are the following lEMP design means the covalent immobilization 

Support External layer of the support Functional group Particle diameter (nm) EIMP CLEMPA EIMP CLEMPA 

---

Figure 4 The different approaches to the production of carrier-free immobilized enzymes (A) crystallization (B) aggregation (C) spray-drying and (D) direct cross-linking. AGG, aggregates CLE, cross-linked dissolved enzyme CLEA, cross-linked enzyme aggregate CLEC, cross-linked enzyme crystal CSDE, cross-linked spray-dried enzyme CRY, crystals SDE, spray-dried enzyme.

Roessl U, Nahalka J, Nidetzky B. Carrier-free immobilized enzymes for biocatalysis. Biotechnol Lett 2010 32 341-50. 

Carrier-free immobilized EHs were prepared using the cross-linked enzyme aggregate (CLEA) technology, as exemplified by the CLEA formation of a mixture of two EHs from mung beans. Compared to the free enzymes, the CLEAs exhibited significantly shorter reaction times and higher enantioconvergence for the hydrolysis of SO in a biphasic n-hexane buffer reaction system, which ensured low levels of nonenzymatic hydrolysis of the substrate [59]. 

Research revealed crosslinked chitosan microspheres to be a good carrier of immobilized enzymes. The covalent attachment of protease inhibitors to chitosan can be achieved via the primary amino groups or the hydroxyl groups of chitosan. Several researches have reported chitosan microspheres as the support to immobilize enzymes [55, 56]. For instance. Wan et al. used chitosan microspheres as the support to immobilize laccase [57]. Chitosan microspheres were added to the laccase solution in phosphate buffer. After 24 h under stirring, the laccase immobilized chitosan microspheres were obtained. Compared with the free enzyme, immobilized laccase of chitosan microspheres showed a lower specific activity but had a similar substrate affinity with improved stability which made it more attractive in the application aspect. Furthermore, they also investigated the reuse of the immobilized laccase. The results showed After being reused fifteen times, the immobilized laccase maintained at least 80% of its initial activity. 

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]. 

Enzymes are an attractive tool in asymmetric catalysis and efficiently complement traditional chemical methods [32,33]. The use of biocatalysts makes it possible to carry out chemical transformations without the need for laborious protection and deprotection steps [34]. Immobilized enzymes are preferred over free enzymes in solution, due to the possibility of repeated use, higher resistance to denaturing effects, and easy separation. The use of a structured support material could be an interesting alternative for conventional particulate enzyme carriers. When optimizing the use of immobilized enzymes, the immobilization method chosen is a very important factor to consider [35]. In this study, a reaction in an organic medium is considered most enzymes do not readily dissolve in organic media, and the enzyme will not detach from the support. This makes physical adsorption a very suitable technique to prepare a biocatalyst for use in an organic medium... 

Cao L, van Rantwijk F, Sheldon R (2000) Cross-linked enzyme aggregates a simple and effective method for the immobilization of penicillin acylase. Oig Lett 2 1361-1364 Cao L, van Langen F, van Rantwijk F et til. (2(X)1) Cross-linked aggregates of peniciUin acylase robust catalysts for the synthesis of (3-lactam antibiotics. J Mol Catal B Enzym 11 665-670 Cao L, van Langen L, Sheldon R (2003) ImmobDised enzymes carrier-bound or carrier-free Cuir Opin Biotechnol 14 1-8... 

Km reflects the affinity between the enzyme and the substrate, and the Km of immobilized enzyme changes little or much, depending on the interaction between immobilized enzyme and carrier. When enzyme is immobilized using carrier binding, due to the electrostatic interaction between the immobilized enzyme and the carrier. Km of the immobilized enzyme decreases. Maximum reaction rate may differ in terms of fixed methods. The maximum reaction rate of the invertase, immobilized by porous glass using covalent binding method is the same as the free enzyme while the maximum reaction rate of the invertase embedded by... 

The storage stability of most immobilized enzyme is greater than that of the free enzyme. The studies on the immobilized CGTase showed that, due to the new bonds between the enzyme and the carriers, the resistance of the immobilized CGTase on the environmental factors increases. 

Despite the advantages of enzymes immobilized on noncatalytic matrices, the yield and productivity of the reaction can be reduced simply due to the presence of the noncatalytic mass of the carrier. There has therefore been much interest in the development of carrier-free systems, in which enzyme molecules are linked to each other to form large complexes. These are inherently immobilized because individual enzyme molecules are no longer free to diffuse in solution, but they are largely undiluted by inert molecules and therefore retain a greater degree of activity than carrier-bound enzymes. This article discusses strategies for enzyme immobilization in carrier-based and carrier-free systems and considers some of their major applications. 

The use of a carrier or polymeric matrix to immobilize enzymes introduces a large noncatalytic component into the system which has the potential to interfere with the catalytic properties of the enzyme and reduce its activity compared to the same mass of free enzyme. Although this disadvantage is balanced by the reusability of immobilized enzymes, it would be even more beneficial if the activities of immobilized enzymes could match those of free enzymes. Carrier-free systems, in which enzyme molecules are linked to each other to form large complexes, may provide a solution to this problem. In a carrier-dependent system, up to 99.9% of the mass is taken up by the noncatalytic matrix. In noncarrier systems, 100% of the complex has the potential to retain catalytic activity. 

Kinetics of the Reactions Catalyzed by Immobilized Enz)mies. The kinetics of the reactions catalyzed by the immobilized enz3mies is influenced by a series of factors inexistent for the free enzymes, such as conformational and steric transformations of the proteic chain induced by the carrier, the diffusion of the substrate and of the reaction products to and from the active centers, the composition of the microsystem created by the carrier. The diffusion effects are reflected by the values of the Michaelis constants (K ). The of an immobilized enzyme is generally higher identical and only in few cases lower than that of the free enz3nne. Higher % values are generally due to diffusion effects. 

Immobilized enzymes are defined as enzymes physically confined or localized in a certain defined region of space with retention of their catalytic activities, which can be used repeatedly and continuously. This definition is applicable to the enzymes as well as aU types of biocatalysts such as cellular organelles, microbial cells, plant cells, and animal cells. In some cases, these biocatalysts are bound to or within insoluble supporting materials (carriers) by chemical or physical binding. In other cases, biocatalysts are free, but confined to limited domains or spaces of supporting materials (entrapment). 

An edited series of papers on the subject of immobilized enzymes for industrial reactors cover the following aspects basic enzymology carriers including controlled-pore glasses for immobilization immobilization by covalent attachment, entrapment, adsorption, and inorganic brid formation comparative characteristics of free and immobilized enzymes inunobilized co-enzjrmes design and operation of immobilized enzyme reactors and applications of immobilized enzymes. ... 

Continuous process possible Improved product quality Re-use of enzyme Product free of enzyme Losses of enzyme activity with immobilizing process Leaching effects Expenses for carrier substances and immobilizing... 

The chemical modification of protein is of importance for a number of reasons. It provides derivatives suitable for sequence analysis, identifies the reactive groups in catalytically active sites of an enzyme, enables the binding of protein to a carrier (protein immobilization) and provides changes in protein properties which are important in food processing. In contrast to free amino acids and except for the relatively small number of functional groups on the terminal amino acids, only the functional groups on protein side chains are available for chemical reactions. 

---