Protein-Coated Polymer as a Matrix for Enzyme Immobilization: Immobilization of Trypsin on Bovine Serum Albumin-Coated Allyl Glycidyl Ether–Ethylene Glycol Dimethacrylate Copolymer Lakshmi Swarnalatha Jasti, Sandhya Rani Dola, Thenkrishnan Kumaraguru, Sreedhar Bajja, and Nitin W. Fadnavis Indian Inst. of Chemical Technology, Uppal Road, Hyderabad, India Uma Addepally Centre for Biotechnology, Jawaharlal Nehru Technological University, Kukatpally, Hyderabad, India Kishor Rajdeo, Surendra Ponrathnam, and Sarika Deokar Polymer Science & Engineering, Chemical Engineering Division, National Chemical Laboratory, Pashan Road, Pune, India DOI 10.1002/btpr.1871 Published online January 22, 2014 in Wiley Online Library (wileyonlinelibrary.com) Allyl glycidyl ether (AGE)–ethylene glycol dimethacrylate (EGDM) copolymer with 25% crosslink density (AGE-25) shows excellent bovine serum albumin (BSA) adsorption (up to 16% (w/w)) at pH 8.0 and the adsorbed BSA is strongly bound. This protein-coated polymer provides a novel matrix with naturally existing functional groups such as thiol, amino, and carboxylic acid that are available for covalent immobilization of functional enzymes. Employing appropriate strategies, trypsin as a model protein was covalently bound to BSA- coated matrix both independently, and in a stepwise manner on the same matrix, with less than 5% loss of enzyme activity during immobilization. Glutaraldehyde crosslinking after immobilization provide stable enzyme preparation with activity of 510 units/g recycled up to six times without loss of enzyme activity. AFM studies reveal that the polymer surface has protein peaks and valleys rather than a uniform monolayer distribution of the protein and the immobilized enzyme preparation can best be described as polymer supported cross- linked enzyme aggregates (CLEAs). V C 2014 American Institute of Chemical Engineers Bio- technol. Prog., 30:317–323, 2014 Keywords: trypsin, bovine serum albumin, immobilization, allyl glycidyl ether, ethylene glycol dimethacrylate Introduction Enzymes immobilized on solid supports have a wide range of applications ranging from production of pharmaceuticals 1– 3 and diagnostic kits 4 to production of food supplements and beverages. 5 Several strategies are employed for immobi- lizing enzymes, which can vary from simple adsorption or entrapment to covalent bond formation with a polymeric support bearing functional groups. 6–10 A special technique of covalent crosslinking of enzymes through bifunctional cross- linker such as glutaraldehyde provides crosslinked enzyme crystals and crosslinked enzyme aggregates (CLEAs), which are very useful in biotransformations. 11 A survey of literature shows that the most popular reactions for covalent coupling of a protein involve the amino, thiol, and carboxylic acid groups, and depending on the choice of functional groups available for reaction, well-defined strategies have been developed for formation of the covalent bond between the enzyme and the support. 12 In our earlier studies on binding of a-chymotrypsin, trypsin, alcohol dehydrogenase, etc., on allyl glycidyl ether (AGE)–ethylene glycol dimethacrylate (EGDM) copolymers, we had observed that the epoxy- activated copolymer was able to bind as much as 25% (w/w) protein, but this was accompanied by a serious loss of enzyme activity (>80%). 13,14 We had to devise a strategy of using reverse micelles to overcome the problem of adsorption-induced denaturation. 15 During these studies, we also observed that the adsorbed proteins were rather strongly bound to the polymer matrix and it was quite difficult to wash out the adsorbed protein. This was quite interesting because the bound proteins possess all the functional groups required for covalent bonding, especially the thiol, amino, and carboxylic acid, all in the same molecule. Indeed, one can choose a functional group for binding of another enzyme on the protein-coated polymer surface, without going through the complicated synthesis of a specially designed functional- ized polymer matrix. In this article, we successfully demonstrate this concept. After screening some of the com- mercially available epoxy-activated polymers, we selected copolymer of AGE–EGDM with 25% crosslink density (AGE-25) for coating with bovine serum albumin (BSA) by simple adsorption at pH 8.0. BSA contains 60 Lys, 41 Asp, 58 Glu, and 35 Cys residues 16 and depending on Correspondence concerning this article should be addressed to N.W. Fadnavis at fadnavisnw@yahoo.com; fadnavis@iict.res.in V C 2014 American Institute of Chemical Engineers 317