Application of Immobilized Enzymes as Pharmaceutical
Chemical immobilization of proteins and enzymes was first attempted in 1960s, and it is an emerging approach to new drug therapies. Immobilization means the enzymes with restricted mobility or rendered less motile by chemical or physical treatment. It was first prepared by loading to polymeric matrices or binding onto carrier materials. Industrial use of enzymes is greatly limited because they are relatively unstable, have a very high cost of purification, and have cumbersome process of recovery of active enzyme from reaction mixture after the completion of catalytic process. Immobilized enzymes are more stable to pH and temperature stress and less susceptible to the denaturing agents. In addition, an immobilized enzyme should have long-term stability and unaltered sensitivity and biological activity after attachment to the matrix than free enzyme when used as therapeutic purpose (Klein and Langer 1986). Immobilization has been successfully utilized for studies with such enzymes, as cytochrome P-450, UDP-glucuronosyltransferases, glutathione S-transferases, S-methyltransferases, and N-acetyltransferases (Dulik and Fenselaut 1998).
One of the major applications of immobilized enzymes in pharmaceutical industry is the production of 6-aminopenicillanic acid (6-APA) by deacylation of the side chain in either penicillin G or V, using penicillin acylase (penicillin amidase). Today more than 50 % of 6-APA is enzymatically produced using the immobilized route which is core of penicillin antibiotic. Penicillin amidase from E. coli is immobilized on cellulose triacetate fibers for producing 6-APA from penicillin G (Alvaro et al. 1990). Similarly for producing 6-APA from penicillin V, penicillin amidase is immobilized by covalent binding to Amberlite XAD-7 with glutaraldehyde through physical adsorption to bentonite or by ionic binding to DEAE-Sephadex and also by covalent binding to a copolymer of acrylamide and maleic anhydride (Arshad et al. 2007). The major reasons for its success is in obtaining a pure product, thereby minimizing the purification costs (Giordano et al. 2006). This process of the immobilized enzyme technology was also approved in India. The first industrial process for the production of 6-APA was started in 1970s by Astra, Sweden, and Riga Biochemical Plant (former USSR).
Immobilization has also been used for the production of 7-aminodeacetoxycephalosporanic acid, an intermediate in the production of semisynthetic cephalosporins. Conversion of 7-amino-3-deacetoxy-cephalosporanic acid (7-ADCA) to cephalexin by immobilized penicillin G acylase (IMPGA) has been investigated. It was observed that under optimized conditions, IMPGA can attain 85 % conversion of 7-ADCA to cephalexin. Furthermore, IMPGA can be reused for about ten cycles (Maladkar 1994). Production of cefazolin by immobilized cefazolin synthetase from E. coli as a biocatalyst has been possible. The complex of the physicochemical studies makes it possible to design a highly efficient technological process for production of cefazolin (Kurochkina and Nys 1999). Macrolide antibiotics tylosin and nikkomycin also can be produced by Streptomycin spp., immobilized with calcium alginate.