Immobilized enzymes are used in organic syntheses to fully exploit the technical and economical advantages of biocatalysts based on isolated enzymes. Immobilization enables the separation of the enzyme catalyst easily from the reaction mixture, and can lower the costs of enzymes dramatically. This is true for immobilized enzyme preparations that provide a well-balanced overall performance, based on reasonable immobilization yields, low mass transfer limitations, and high operational stability. There are many methods available for immobilization which span from binding on prefabricated carrier materials to incorporation into in situ prepared carriers. Operative binding forces vary between weak multiple adsorptive interactions and single attachments through strong covalent binding.Which of the methods is the most appropriate is usually a matter of the desired applications. It is therefore the intention of this paper to outline the common immobilization methods and reaction technologies to facilitate proper applications of immobilized enzymes.

Immobilized Enzymes: Methods and Applications

Trypsin immobilization onto continuous "molded" rods of porous poly(glycidyl methacrylate-co-ethylene dimethacrylate) and some applications of the conjugate have been studied. The rods polymerized within a tubular mold (chromatographic column), were treated in situ with ethylenediamine, activated with glutaraldehyde and finally modified with trypsin. The performance of the trypsin-modified rods was evaluated and compared to that of poly(glycidyl methacrylate-co-ethylene dimethacrylate) beads, modified with the same enzyme. Overall the enzyme-modified rods performed substantially better than the corresponding beads. In particular, the performance of the molded supports as enzymatic reactors or as chromatographic media benefits greatly from the enhanced mass transfer that is characteristic of the molded rod at high flow rates. (c) 1996 John Wiley & Sons, Inc.

Immobilization of trypsin onto "molded" macroporous poly(glycidyl methacrylate-co-ethylene dimethacrylate) rods and use of the conjugates as bioreactors and for affinity chromatography.

Use of monolithic supports for enzyme immobilization has rapidly expanded since we published the preceding paper in the series of articles concerned with this topic almost three years ago. Many groups worldwide have realized the benefits of applying monoliths as support structures and used a variety of techniques to immobilize many different enzymes. Although some of these new developments are just refinements of the methods developed previously, some notable new approaches have also been reported. This review summarizes the literature published since 2006 and demonstrates the broad variability of reactive monoliths prepared from silica as well as from organic polymers in the form of disks, columns, and capillaries. All these monoliths were prepared by direct formation from reactive precursors or activation of preformed inactive structures. Interestingly, most of the applications of monolithic enzyme reactors target proteolytic digestion of proteins for proteomic analysis.

Less common applications of monoliths: IV. Recent developments in immobilized enzyme reactors for proteomics and biotechnology.

Publisher Summary This chapter discusses a survey of enzyme coupling techniques and enzyme immobilization, with emphasis on the carder matrices. Immobilized enzymes have been used to prepare enzyme electrodes and transistors for diagnostic purposes, as supports in high-performance liquid chromatography (HPLC) separations and as therapeutic agents, for chemical synthesis. The majority of immobilized enzymes used in these applications have been prepared using a small group of matrices. The most prevalent and most widely applicable of the commercially available matrices for covalent coupling enzymes are the polysaccharides, agarose and cellulose, and their derivatives. The most widely employed method of immobilizing antibodies in solid phase, heterogeneous, immunoassay is absorption on polystyrene, either as beads or as styrene tubes. However, no single immobilization method is best for all enzymes or all applications of any given enzyme.

[2] A survey of enzyme coupling techniques

This review summarizes the recent contributions to the rapidly growing area of immobilized enzymes employing both silica and synthetic polymer-based monoliths as supports. Focus is mainly on immobilized proteolytic enzyme reactors designed for studies in proteomics. Porous monoliths emerged first as a new class of stationary phases for HPLC in the early 1990s. Soon thereafter, they were also used as supports for immobilization of proteins and preparation of both stationary phases for bioaffinity chromatography and enzymatic reactors. Organic polymer-based monoliths are typically prepared using a simple molding process carried out within the confines of a "mold" such as chromatographic column or capillary. Polymerization of a mixture comprising monomers, initiator, and porogenic solvent affords macroporous materials. In contrast, silica-based monoliths are first formed as a rigid rod from tetraalkoxysilane in the presence of PEG and subsequently encased with a plastic tube. Both types of monolith feature large through-pores that enable a rapid flow-through. Since all the solutions must flow through the monolith, the convection considerably accelerates mass transfer within the monolith. As a result, reactors including enzyme immobilized on monolithic support exhibit much higher activity compared to the reactions in solution.

Less common applications of monoliths: I. Microscale protein mapping with proteolytic enzymes immobilized on monolithic supports.

Two types of bead-form macroporous carriers based on glycidyl methacrylate with ethylene dimethacrylate copolymers were used for the immobilization of penicillin amidase either directly or after chemical modification. Direct binding through oxirane groups, which is equally efficient at pH 4.2 and 7, is relatively slow and brings about an activity loss at low enzyme concentrations. The most efficient immobilization was achieved on glutaraldehyde-activated amino carrier, irrespective of whether the amino groups were formed by ammonia or 1,6-diaminohexane treatment of the original oxirane carrier. Hydrazine treatment gave lower immobilization yields. The same is true of the azide method independent of the length of the spacer. Most enzyme activity was preserved by coupling the carbodiimide-activated enzyme to the carrier with alkyl or arylamino groups at the end of a longer substituent. Immobilization on diazo-modified carrier gave average results. Rapid immobilization by a lysine-modified phosgene-treated carrier resulted in an activity loss. It is suggested that multipoint and very tight attachment of the enzyme molecule to the matrix decreased the activity. The immobilized activity is quite stable in solution and very stable upon lyophilization with sucrose.

J. Drobnik

Papers

Enzyme immobilization techniques on poly(glycidyl methacrylate-co-ethylene dimethacrylate) carrier with penicillin amidase as model.