Cross-linking, enzyme stabilization method

Gupta, M.N. and S. Raghava, Enzyme stabilization via cross-linked enzyme aggregates. Methods Mol. Biol., 2011. 679 133-145. 

Immobilization by chemical cross-linking without the addition of an inert carrier or matrix can provide the means to stabilize and reuse a biocatalyst without dilution of volumetric activity. A major deficiency in all of the aforementioned immobilization methods is that a substantial amount of a catalytically inert carrier or matrix is used to bind or contain the biocatalyst. In many cases, the amount of carrier is two orders of magnitude higher than the protein catalyst. Unfortunately, direct cross-linking of the enzyme, followed by precipitation of an amorphous solid often results in low activity and poor mechanically properties and so this method is not often used. Recently, however, cross-linked enzyme crystals have been reported to give many of the desirable properties of immobilized enzymes without the need for a support material (Sect. 6.4.1). 

Another category of enzymatic transformations in multiphase systems is enzymes immobilized on the reactor wall as presented in Table 10.4. Enzymes are advantageously used in immobilized form because this strategy allows for increased volumetric productivity and improves stability. Continuous mode of operation is employed in these systems. The approaches commonly used for immobilization in conventional multiphase biocatalysis can also be employed in microreactors such as covalent methods, cross-linked enzyme aggregates (CLEA), and adsorption methods. The experimental setups can either be chip-type reactors with activated charmel surface walls where enzyme binds, or enzyme immobilized monolith reactors, where a support is packed inside a capillary tube. 

Rev. 42 (2013) 6213-6222. (d) L. Cao, F.v. Rantwijk, R.A. Sheldon, Cross-linked enzyme aggregates a simple and effective method for the immobilization of penicillin acylase, Org. Lett. 2 (2000) 1361-1364. (e) T. Matsuda, K. Nakayama, T. Abe, M. Mukouyama, Stabilization of pyruvate decarboxylase under pressurized carbon dioxide and water biphasic system, Biocatal. Bio-trans. 28 (2010) 167-171. (f)T. Matsuda, R. Marukado, M. Mukouyama, . Harada, K. Nakamura, Asymmetric reduction of ketones by Geotrichum candidum immobilization and application to reactions using supercritical carbon dioxide. Tetrahedron Asymm. 19 (2008) 2272-2275. 

Because enzymes can be intraceUularly associated with cell membranes, whole microbial cells, viable or nonviable, can be used to exploit the activity of one or more types of enzyme and cofactor regeneration, eg, alcohol production from sugar with yeast cells. Viable cells may be further stabilized by entrapment in aqueous gel beads or attached to the surface of spherical particles. Otherwise cells are usually homogenized and cross-linked with glutaraldehyde [111-30-8] to form an insoluble yet penetrable matrix. This is the method upon which the principal industrial appHcations of immobilized enzymes is based. 

Tor [7] developed a new method for the preparation of thin, uniform, self-mounted enzyme membrane, directly coating the surface of glass pH electrodes. The enzyme was dissolved in a solution containing synthetic prepolymers. The electrode was dipped in the solution, dried, and drained carefully. The backbone polymer was then cross-linked under controlled conditions to generate a thin enzyme membrane. The method was demonstrated and characterized by the determination of acetylcholine by an acetylcholine esterase electrode, urea by a urease electrode, and penicillin G by a penicillinase electrode. Linear response in a wide range of substrate concentrations and high storage and operational stability were recorded for all the enzymes tested. 

Despite these improvements, there are other important biosensor limitations related to stability and reproducibility that have to be addressed. In this context, enzyme immobilisation is a critical factor for optimal biosensor design. Typical immobilisation methods are direct adsorption of the catalytic protein on the electrode surface, or covalent binding. The first method leads to unstable sensors, and the second one presents the drawback of reducing enzyme activity to a great extent. A commonly used procedure, due to its simplicity and easy implementation, is the immobilisation of the enzyme on a membrane. The simplest way is to sandwich the enzyme between the membrane and the electrode. Higher activity and greater stability can be achieved if the enzyme is previously cross-linked with a bi-functional reagent. 

Both chemical and physical methods may be used to immobilize biocatalysts while retaining or modifying their activity, selectivity, or stability. Among the techniques used for immobilization of enzymes are physical adsorption, covalent bonding, ionic binding, chelation, cross-linking, physical entrapment, microencapsulation, and retention in permselective membrane reactors. The mode of immobilization employed for a particular application depends not only on the specific choice of enzyme and support, but also on the constraints imposed by the microenvironment associated with the application. 

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