Activity Studies of Immobilized Subtilisin on Functionalized Pure Cellulose-Based Membranes Jiangling Liu, † Jianquan Wang, ‡ Leonidas G. Bachas, ‡ and Dibakar Bhattacharyya* ,† Department of Chemical and Materials Engineering and Department of Chemistry, University of Kentucky, Lexington, Kentucky 40506 The activity of immobilized subtilisin BPN′ on pure cellulose-based membrane support was investigated using site-directed and random immobilization approaches. The catalytic activity of site-directed immobilized subtilisin on pure cellulose fiber-based materials was found to be 81% of that in homogeneous solution, while that of randomly immobilized subtilisin was 27%. Pure cellulose membrane supports provided large surface areas for high enzyme loading without diffusional limitations. The activity of immobilized subtilisin on pure cellulose support was more than twice that on a modified polyether sulfone (MPS) membrane, which was attributed to the higher hydrophilicity of cellulose. Immobilized subtilisin maintained its initial activity for 14 days at 4 °C and 7 days at 24 °C. The immobilized enzyme could resist higher temperature and operate over a wider range of pH without loss of activity. This study showed that pure cellulose fiber-based membranes are well suited for enzyme immobilization and biocatalysis. 1. Introduction The application of enzyme catalysts in industrial processes has drawn increased attention in recent years. Unlike conventional catalysts, enzymes can be used to catalyze reactions at ambient temperature and atmo- spheric pressure with high selectivity and efficiency (1, 2). The applications of enzyme-catalyzed reactions in homogeneous solution are limited as a result of the need for separation of the enzyme from the reaction mixture, possible contamination of the products, and the loss of catalytic activity. For immobilized enzyme systems, the elimination of enzyme separation steps and the enhance- ment of enzyme stability make enzyme-based industrial applications more advantageous (3). There are many physical approaches for enzyme im- mobilization, including adsorption on solid supports, entrapment into gel matrices, and microencapsulation with semipermeable membranes (4). Immobilization through chemical bonding between a support matrix and an enzyme has several advantages over physical methods (5). For example, covalently bound enzymes are usually more stable during storage and less likely to leach out of the immobilization matrix during continuous operation processes. The chemical and physical properties of enzymes are often changed after immobilization. The biggest disad- vantage of immobilized enzymes compared with those in homogeneous solution is the loss of catalytic activity (6). Therefore, it is important to choose an appropriate immobilization method as well as a suitable immobiliza- tion support matrix to minimize the loss of catalytic activity of immobilized enzymes. The orientation of the active site of an immobilized enzyme also plays an important role on its activity. Enzymes can be randomly or site-directly immobilized on a solid support (7, 8). In random immobilization, the enzyme is usually bound to the support via multiple amino acid groups on the enzyme. The large number of bonds may result in deformation of the active site of the enzyme and hence reduce the enzyme activity. The activity of an immobilized enzyme can be increased greatly by site-directed immobilization in which the enzyme is attached to the support through a single bond formed between the support and a reactive residue of the enzyme at a site away from its active site. For example, Huang et al. (9) have produced mutant subtilisin with serine 145 replaced by cysteine through site-directed mutagenesis, which was subsequently site-directly im- mobilized onto thiol-activated surfaces. This study dem- onstrated that the site-directly immobilized subtilisin has higher activity than the randomly immobilized enzyme. The physical and chemical properties of the support matrix also play an important role in the activity of immobilized enzymes (10-14). For example, investiga- tions of immobilized enzymes on hydrophobic supports, such as modified polyether sulfone (MPS) membranes, have shown that most of the enzyme activity was lost after immobilization (13, 14). Additionally, it has been demonstrated that the activity of immobilized enzymes increases as the hydrophilicity of the immobilization support increases (11, 13). High enzyme loading and fast reaction rate are es- sential to achieve high productivity in industrial applica- tions involving enzymes. An immobilization matrix with a large internal surface area (BET surface area) is desirable for increasing enzyme loading and maximizing immobilized enzyme catalytic activity. Membranes and other porous materials provide high surface area for enzyme immobilization, although they may cause diffu- * Ph: 859-257-2794. Fax: 859-323-1929. E-mail: db@ engr.uky.edu. † Department of Chemical and Materials Engineering. ‡ Department of Chemistry. 866 Biotechnol. Prog. 2001, 17, 866-871 10.1021/bp010065l CCC: $20.00 © 2001 American Chemical Society and American Institute of Chemical Engineers Published on Web 07/21/2001