Enzymes can be immobilized onto solid media using nonspecific hydrophobic adsorption or ionic-bonding techniques. Noncovalent immobilization on Amberlite or Duolite resins is typically done using neutral, or near-neutral, pH conditions. The acrylic ester chemistry of the Amberlite XAD7HP resin and phenol-formaldehyde chemistries of Amberlite XAD761 and Duolite A7 resins provide gentle binding, which can lead to improved activity of the bound enzyme compared to other supports. Duolite A7 resin is particularly effective for the immobilization of b-glycosidases.
More hydrophobic chemistries such as the polystyrenic Amberlite XAD16 resin can be modified by grafting acrylate polymer on the adsorbent, creating a more hydrophilic surface for enzyme attachment. Nonmodified polystyrenic resins such as Amberlite XAD1180 can be used directly for enzyme adsorption in processes to produce enantiomerically pure compounds. With adsorption, enzyme leakage from the support is sometimes problematic. Gluteraldehyde, however, can be used for either preimmobilization activation of the resin or post-immobilization crosslinking of the immobilized enzyme improves binding stability.
Amberlite IRC50 resin (the precursor to Amberlite FPC3500 resin) is a cation exchange resin that has been used for ionic immobilization of enzymes such as fructozyme L, lipase, and phospholipase D. For b-galactosidase, Amberlite IRC50 is first reacted with polyethyleneimine and then reacted with gluteraldehyde. The gluteraldehyde acts as a crosslinker for the polyethyleneimine and provides a spacer arm for enzyme attachment.
An example of high-capacity binding of enzymes to nonmodified Amberlite IRC50 resin is shown in the Figure. Here, two different enzymes are bound to IRC50 using buffered conditions at a pH of 7.3 and contact times ranging from 4 to 12 hours. The data demonstrates that basic enzymes such as cytochrome C (pI of 9.6) and lysozyme (pI of 10.0) are well adsorbed on Amberlite IRC50 resin.
Another form of ionic attachment employs the use of anion exchange materials. Duolite A568 and Duolite A561 resins provide significant immobilization yields and protein loading, however, the immobilized enzyme specific activity can vary depending on the enzyme. Enzyme stability can improve with immobilization. For example, N-carbamyl-d-amino acid amidohydrolase demonstrates superior heat stability when immobilized on Duolite A568 resin.