Covalent Enzyme Immobilization onto Photopolymerized Highly Porous Monoliths

Abstract

In recent years enzymes have found widespread use in areas as diverse as chemical synthesis, decontamination of waste streams, and biosensors. For ease of application and for stabilization purposes, enzymes are often immobilized on solid supports. These supports can include inorganic substrates such as silicon or glass, as well as organic materials such as polymers or hydrogels. In current industrial processes, enzymes are often simply adsorbed onto the material. Although this is a cost-effective method for supporting enzymes, these biocatalytic materials often suffer from decreased activity after prolonged usage because of the leakage of adsorbed enzymes during catalysis and recycling. Furthermore, since there is a lack of control over the enzyme positioning, only a fraction of the enzyme is in contact with the environment and therefore used effectively. In more sophisticated applications, such as sensors and analytical devices, covalent immobilization is achieved using carefully designed anchors and surface modification. These multistep procedures lead to welldefined systems, but costs are raised considerably and these methods are difficult to extend to large-scale applications. In this article we describe a simple and effective strategy for immobilizing enzymes covalently onto a solid porous support, and ascertain that a large fraction of the enzyme is available for catalysis. To achieve this, a support with a high surface to volume ratio is employed, namely a polymerized high internal phase emulsion (polyHIPE). N-Hydroxysuccinimide esters are introduced into this highly porous monolithic support for covalent coupling with the lysine residues present in proteins. When the activity of the well-known lipase Candida antarctica Lipase B (CAL-B) immobilized on polyHIPE was compared with the commercially available standard, Novozyme 435, a remarkable increase in both activity and stability was observed. PolyHIPEs are highly porous polymers obtained by polymerizing the continuous phase of a HIPE. The emulsion must have a droplet volume fraction of at least 0.74, and can exceed 0.99. The stability of this emulsion is mainly dependent on the nature of the surfactant, which should be soluble only in the continuous phase. PolyHIPEs have a well-defined open cellular morphology, with interconnecting holes (windows) between voids resulting from the continuous phase shrinkage during polymerization (see Fig. 1). Their unique morphology differentiates them from other porous polymers (e.g. gasblown foams), and yields excellent flow-through properties. Since the development of polyHIPEs in the 1980s, numerous applications have been developed, such as solid-phase peptide synthesis supports, catalyst supports, superabsorbents, ion-exchange resins, monolithic supports for cells, and membrane filters. However, the use of polyHIPEs as covalently immobilized enzyme supports for biocatalysis has not yet been reported. C O M M U N IC A TI O N S