Invertase-Lipid Biocomposite Films:Preparation, Characterization, and Enzymatic Activity Sumant Phadtare, † Virginia D’Britto, ‡ Archana Pundle, ‡ Asmita Prabhune,* ,‡ and Murali Sastry* ,† Materials Chemistry and Biochemical Sciences Divisions, National Chemical Laboratory, Pune 411 008, India The formation of biocomposite films of the industrially important enzyme invertase and fatty lipids under enzyme-friendly conditions is described. The approach involves a simple beaker-based diffusion protocol wherein invertase diffuses into the cationic lipid octadecylamine during immersion ofthe lipid film in the enzyme solution. Entrapment of invertase in the octadecylamine film is highly pH-dependent, under- lining the role of attractive electrostatic interactions between the enzyme and the lipid in the biocomposite film formation. The kinetics of formation of the enzyme-lipid biocomposites has been studied by quartz crystal microgravimetry (QCM) measure- ments. The stability of the enzyme in the lipid matrix was confirmed by fluorescence spectroscopy and biocatalytic activity measurements. The biocatalytic activity of the invertase-lipid biocomposite films was comparable to that of the free enzyme in solution and showed marginally higher temperature stability. Particularly exciting was the excellent reuse characteristics of the biocomposite films, indicating potential industrial application of these films. Introduction Studies on protein-lipid interactions are important for technological and biomedical applications such as pro- tocols for drug delivery and immunosensing systems (1- 3). Moreover, the interactions of different functional groups on the protein surface with lipid molecules has helped researchers address a number ofundamental issues related to protein orientation, accessibility,and action in biological membranes (4). For obvious reasons, enzymes have received considerable attention, and a number of immobilization protocols developed. Immobi- lized biocatalysts are advantageous for commercial ap- plications because ofconvenience in handling,ease of separation ofenzymes from the reaction mixture and reuse, low product costs, and possible increase in thermal and pH stability (5). Encapsulation also protects the enzymes against degradation, aggregation,and deami- dation while rendering the enzymes accessible to sub- strates and cofactors for biosensing and biocatalytic applications (6, 7). An important requirement for im- mobilizing the proteins is that the matrix should provide a biocompatible and inert environment, i.e., it should not interfere with the native structure of the protein and thereby compromise its biological activity. Consequently, a number of processes for enzyme im- mobilization in silica nanotubes (8), within phospholipid bilayers (9), on self-assembled monolayers (10), in Lang- muir-Blodgett films (11), within polymer matrix (12) and galleries of R-zirconium phosphate (13), mesoporous materials (14),and in thermally evaporated fatty lipid films (15) have been developed, each with its character- istic pros and cons. In this laboratory, some of us have reported the use of thermally evaporated fatty lipid films in the formation of protein-lipid biocomposites, wherein proteins such as cytochromec/hemoglobin (16)and enzymes such as pepsin (17), fungal protease (18), endoglucanase (19), and penicillin G acylase (20) have been studied.The utility of such thermally evaporated lipid films in the generation of patterned protein as- semblies has also been demonstrated (21). The process for the formation of such biocomposites is primarily based on electrostatic interactions between the lipid matrix and charges on the protein surface. Depending on the pI of the protein/enzyme, both cationic (e.g., octadecylamine, ODA) (16, 17, 19) and anionic (e.g., stearic acid, StA) (16, 18, 20) lipids may be used for immobilizing proteins. We have observed that hydrophobic and hydrogen bonding interactions also play a role in the biocomposite formation process (16-20). In this paper we report on the formation of invertase-lipid biocomposites by a simple diffusion process under enzyme-friendly conditions as shown in Scheme 1, steps a and b. Our protocol consists of thermal evaporation of a thin film of the cationic lipid octadecyl- amine (ODA), which is then immersed in invertase solution. During immersion, invertase diffuses into the ODA host and is entrapped electrostatically.The im- mobilized enzyme showed excellent specific activity and temperature stability. Coupled with the excellent reuse characteristics over five successive reaction cycles, the formation of invertase-ODA biocomposite films repre- sents an important addition to the lipid-based enzyme immobilization strategy developed in this laboratory. Presented below are details of the investigation. Experimental Section Chemicals. Invertase (â-fructofuranosidase, E.C.3.2.1.26.)was obtained from BDH (British Drug House). Octadecylamine (ODA, CH 3 (CH 2 ) 17 NH 2 ), sucrose, * To whom correspondenceshould be addressed. E-mail: sastry@ems.ncl.res.in; asmita@dalton.ncl.res.in. † Materials Chemistry Division. ‡ Biochemical Sciences Division. 156 Biotechnol. Prog. 2004, 20, 156 − 161 10.1021/bp034236t CCC:$27.50 © 2004 American Chemical Society and American Institute of Chemical Engineers Published on Web 10/25/2003