Immobilized Enzymes

The large cavities of Cavilink polymers are ideally suited for immobilization of large molecules such as enzymes. The micrometer-size cavities allow proper and efficient covalent attachment of enzymes to the interior walls of the polymers. [Click on "The Polymers" tab at left for a full description of the polymers and their properties.]  In addition, these cavities provide clear access to bound enzymes by substrates without steric hindrance associated with conventional solid supports. The interconnecting pores of Cavilink permit ready egress of reaction products. Enzyme-substrate reactions are more efficient, and enzyme activity is much longer than with conventional polymers. This was demonstrated by Hsuanyu et al. in 2007. and is available at: Immobilized Enzymes 2007. A publication providing some applications of immobilized enzymes is found at: Immobilized Enzyme Applications. A monograph prepared in 2008 illustrates more recent developments in this field: Immobilized Enzymes 2008.

Enzyme stability

 Stability of β-glucosidase immobilized onto Cavilink polymers was investigated and compared with commercial polymers, IRA-900 and XAD-2. Figure 1 summarizes the results. (Details of the study are found elsewhere. [1]) The figure shows rapid loss of enzyme activity for the commercial polymers. Cavilink polymers containing immobilized β-glucosidase maintained about 90% enzyme activity for twelve months.

 This remarkable feature appears to be related to the large, interconnected cavities of Cavilink. It is speculated that much of the enzyme initially loaded onto the commercial polymers was not covalently bound to the surface due to steric hindrance associated with terminating, irregular pores. Thus, much of the unbound enzyme diffused from the commercial polymer over time, resulting in loss of activity.

 In contrast, the open, interconnected cavities of Cavilink polymers allowed full access to regions of enzymes to be bound so that covalent binding was very efficient. This resulted in exceptional stability of the combination product (enzyme bound to Cavilink polymer).  

 Immobilized enzyme reactivity

 To compare reaction rates of immobilized enzyme with free enzyme in solution, anti-horse IgG conjugated peroxidase was bound to a Cavilink polymer. This was allowed to react with urea hydrogen peroxide and orthophenylenediamine (OPD). Reactivity of the combination product (enzyme bound to Cavilink) was compared with the free enzyme in solution. The results are shown in Figure 2. (Details of the study are found elsewhere.[2] )

 As seen in the figure, reaction rates of the combination product and free enzyme were identical within experimental error. This is due to the unique open and interconnected cavities of Cavilink. When the enzyme is bound to the polymer, the large cavities permit proper orientation during attachment. When the combination product is allowed to mix with substrate, the interconnected cavities allow rapid ingress and egress. This unique feature of enzymes bound to Cavilink polymers illustrates the exceptional versatility and utility of combination products.   

Binding Capacity 

The open, interconnected structure of Cavilink polymers permits optimum binding of biomolecules. Ideally, enzymes or other large molecules should be bound covalently to ensure stability of the combination product. When conventional macroporous polymers are used in these applications, much of the applied compound is adsorbed and not bound covalently. This occurs due to steric limitations and inability of the tortuous pore structure of these conventional polymers to permit proper orientation of the biomolecule. Consequently, much of the applied compound will diffuse from the matrix (see Figure 1).

 Figure 3 illustrates the stability of a combination product comprised of Cavilink bound with albumin. A cross-linked polystyrene Cavilink structure was aminated and then allowed to react with albumin. The amount of bound albumin was calculated and the combination product was subsequently exposed to a variety of washing methods to determine if any albumin was adsorbed and not covalently bound. Adsorbed protein would be expected to be removed by some of the washes.   

 The effectiveness of Cavilink combination products when used in applications to purify and collect other molecules was investigated. In this experiment, aminated Cavilink was first reacted with protein A to produce a combination product. This material was exposed to IgG and the amount of IgG bound to the combination product was determined. The results are shown in Figure 4.   

It is speculated that the increased binding capacity exhibited by Cavilink combination products is due to the very large micrometer sized cavities and the interconnections that allow large molecules ingress and egress. Once these biomolecules traverse the cavities, proper orientation for binding to the interior polymer wall and subsequent proper alignment with any substrates are achieved.

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