Enzymes immobilization by adsorption

There are two basic procedures for enzyme immobilization by adsorption. Both share the same first steps preparation of the immobilization matrices (discussed above) and the aqueous enzyme solution. Typically, the aqueous solution is fairly concentrated in enzyme (approximately 5 to 40 mg/ml) and buffered (approximately 10 mM) to the optimal pH value of the enzyme. In addition, necessary co-factors, such as NAD(P)+ or NAD(P)H, should be included. Also, the presence of albumin or high-molecular-weight PEG at concentrations of 2 mg per g of matrix have been shown effective in protecting enzymes during the water removal stage of immobi-lization. °° Furthermore, the presence of sorbitol can increase activity and reduce side reactions, such as hydrolysis. -It is recommended that the aqueous solutions be centrifuged prior to use to remove any nonsolubilized matter. 

The adsorption of an enzyme onto a support or film material is the simplest method of obtaining an immobilized enzyme. Basically, the enzyme is attached to the support material by noncovalent linkages and does not require any preactivation step of the support. The interactions formed between the enzyme and the support material will be dependent on the existing surface chemistry of the support and on the type of amino acids exposed at the surface of the enzyme molecule. Enzyme immobilization by adsorption involves, normally, weak interactions between the support and the enzyme such as ionic or hydrophobic interactions, hydrogen bonding, and van der Waals forces (see Figure 44.1). ... 

An example of enzyme immobilized by adsorption was described by Gao and others, who developed a PDMS microreactor for proteolytic digestion with online ESI-MS identification [89]. Trypsin was adsorbed in a poly(vinylidine fluoride) porous membrane. Peptide identification for Cyt< was reported using as little as 0.04 pmol. 

---