Carrier-free enzyme

It may be difficult to conserve initial velocities over a sufficient period of time to warrant appropriate sensitivity of the assays, especially with unactivated enzyme preparations. With carrier-free bilindiin substrate, decrease of the initial velocity may occur earlier than with albumin-solubilized bilirubin ( 116). Spontaneous activation (W7) of enzyme... 

For lipase, initial activity corresponds to the amount of protein that was adsorbed. Specific activity is constant at 1 mmoFs gE for this carrier-enzyme system, which compares to 27% of the free enzyme activity. The trypsin system shows a lower specific activity that is only 10% of the free enzyme. The reason for the lower recovered activity of this system is not known. To rule out possible internal diffusion limitations, the Wheeler-Weisz modulus was estimated, assuming a carrier layer thickness of 0.1 mm for all carriers. Using the data of the experiments performed at 150 rpm, one finds ... 

Cao L, van Langen L, Sheldon RA (2003) Immobilised enzymes carrier-bound or carrier-free Curr Opin Biotechnol 14 387-394... 

Cao, L., Van Langen, L., and Sheldon, R. 2003. Immobilised enzymes Carrier bound or carrier-free Current Opinion in Biotechnology, 14 387-94. 

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]. 

On the other hand, strongly coordinating ionic hqnids, snch as [bmim+][dca ] are potentially nsefnl solvents due to its high solvent capability [3, 51, 52]. For that reason, enzymes derivatives compatible with this kind of ionic liquids have been developed. In this context, CaLB, adsorbed and cross-linked on a polypropylene carrier, maintained its transesteriflcation activity in the ionic liquids [bmim ][N03 ] and [bmim+][dca ], which deactivate the free enzyme [3]. 

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... 

The enzymes must, of course, be linked at some distance from the active site of the enzyme. Such insolubilized enzymes retain their activities, but their immobility may reduce the reaction rate. The properties of such covalently linked enzymes are not always the same as those of the free enzyme. The anionic or cationic nature of the carrier may alter the pH optimum for the reaction (Goldstein, 1969). The binding of the enzyme to carrier may result in steric hindrance and impose restrictions on the specificity of the bound enzyme. This is most... 

Cao L, Eischer A, Bornscheuer UT et al. (1997) Lipase-catalysed solid phase synthesis of sugar fatty acid esters. Biocatal Biotransfor 14 269-283 Cao L, van Langen L, Sheldon, R (2(X)3) Immobilised enzymes carrier-bound or carrier-free Curr Opin Biotechnol 14 1-8... 

Consider those methods that include the chemical binding of the enzyme molecules to an inert carrier (carrier-bound), where linkage can be covalent or non-covalent, and those in which the enzyme protein molecules are chemically linked among themselves, usually through a bifunctional reagent, without the participation of an inert carrier (carrier-free). 

Immobihzation of enzyme is the process where the free enzyme s catalytic activity is completely or largely restricted to a certain space, and is divided into embedded-type and bound-type. For embedded-type, enzyme itself (Fig. 2.22) does not bind, but is embedded in the monomer of the polymer, including plastic, microcapsule and liposome. In the case of bound-type, the enzyme is bound up to combine through the interaction between the enzyme and the carrier, and according to the different forms of combination, it can be divided into adsorption, covalent binding and crosslinking. In the immobihzation of history of CCTase, including almost all of the above methods. 

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. 

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.

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