Activity of Candida rugosa Lipase Immobilized on γ-Fe 2 O 3 Magnetic Nanoparticles Ansil Dyal, ²,‡ Katja Loos, ²,‡ Mayumi Noto, ² Seung W. Chang, ²,‡ Chiara Spagnoli, ² Kurikka V. P. M. Shafi, ²,‡ Abraham Ulman,* ,²,‡ Mary Cowman, ² and Richard A. Gross ² Department of Chemical Engineering, Chemistry and Material Science, Polytechnic UniVersity, 6 Metrotech Center, Brooklyn, New York 11201, and The NSF Garcia MRSEC for Polymers at Engineered Interfaces Received September 30, 2002 ; E-mail: aulman@duke.poly.edu or aulman@photon.poly.edu The use of nanophase materials offers many advantages due to their unique size and physical properties. Hybrid nanoscale materials are well established in various bioprocesses such as nucleic acid detachment, 1 protein separation, 2 and immobilization of enzymes. 3 An important area of interest is the immobilization of proteins and enzymes on magnetic particles. Several magnetic particles 3,4 and magnetic supports such as microspheres of various biomaterials 5 encapsulating the magnetic particles, and copolymers 5 with magnetic particles have been used with good results. However, due to size constraints (usually 75-100 μm), these microparticles cannot be placed at specific locations that are relevant to cellular biochemical processes. Preferably, such particles would possess a very low magnetic hysteresis and high stability. The functionalized γ-Fe 2 O 3 magnetic nanoparticles used as a support in this communication possess all these traits. In addition, because of the inherent structure and size of these particles, they are superparamagnetic which addresses aggregation and flocculation concerns. 6 Here, we report the stability and enzymatic activity of Candida rugosa lipase (E.C.3.1.1.3) immobilized on γ-Fe 2 O 3 magnetic nanoparticles. Lipases are frequently employed enzymes as they are commonly used for the synthesis of enantioenriched monomers and macromers and for polymerization reactions. 7 It is shown here that these enzymes, when immobilized on magnetic γ-Fe 2 O 3 nanoparticles, can be easily separated from the reaction medium, stored, and reused with consistent results. This system offers a relatively simple tech- nique for separating and reusing enzymes over a longer period than that for free enzymes alone and for enzymes which are immobilized by physisorption. This can be explained by the use of covalent im- mobilization that does not permit the loss of enzyme by desorption from the support and protects the enzyme from denaturation by constraining it to the local environment of the particle. 8 However, while the ability to stabilize and recover the enzyme is achieved by chemical immobilization, some enzymatic activity may be lost by chemical bonding since the active site is hidden or restricted from assuming the conformation needed to initiate the catalysis. 9 For the immobilized enzyme in the present case separation is facilitated by the use of a magnet where either the substrate solution is removed while the immobilized enzyme is held in place with a magnetic field or vice versa. γ-Fe 2 O 3 nanoparticles were prepared by sonication of Fe(CO) in decalin and the subsequent annealing of the amorphous Fe 2 O 3 nanoparticles. 10 The average size of the γ-Fe 2 O 3 nanoparticles is 20 ( 10 nm (see Figure 2), with saturation magnetization value of 61emu/g. 10 Figure 1 presents the strategy used to immobilize Candida rugosa lipase on the γ-Fe 2 O 3 nanoparticles. 11-Bromoun- decanoic acid was covalently linked to the nanoparticle surfaces by heating the nanoparticles in acid solution in ethanol by using microwave irradiation for 10 min. Nucleophilic substitution using 2-thiophene thiolate resulted in thiophene-functionalized nanopar- ticles. These were either acetylated using acetic anhydride with iodine as catalyst or reacted with nitrosonium tetrafluoroborate in methylene chloride to produce the nitroso derivative. Given the chemistry of thiophene, we assume that in both cases electrophilic substitution occurred at the 5-position. In all cases full coverage of the surface area was achieved which was proven by thermo- gravimetrical analysis. Notice that coating of the γ-Fe 2 O 3 with 11- bromoundecanoic acid decreases the magnetization of the bare γ-Fe 2 O 3 on average only by 12%, resulting in nanoparticles with value still far greater than any previously reported magnetic support. The acetylated nanoparticles were reacted directly with the enzyme, which was chemically bonded to the nanoparticle surface via a CdN bond. The nitroso-functionalized nanoparticles were reduced to the corresponding amine-functionalized nanoparticles with SnCl 2 , and the enzyme was chemically connected using glutaraldehyde. For the immobilization on acetylated nanoparticles, 17.3 mg of lipase (ca. 8% protein content according to protein assay) was reacted with 32.38 mg of the acetylated nanoparticle in 8 mL of phosphate buffer (10 mM, pH 7.5) under gentle shaking for 24 h at room temperature. The immobilization on amino-functionalized nanoparticles was conducted in a one- or two-pot reaction, respectively. In the one-pot approach, 30 mg of amino-fuctionalized nanoparticles was mixed with 5 μL of a 50 wt % solution of glutaraldehyde in water and 2 mL of phosphate buffer and was shaken for 1 h at 25 °C. Thereafter, 92 mg of lipase was added in ² Polytechnic University. ‡ The NSF Garcia MRSEC for Polymers at Engineered Interfaces. Figure 1. The strategies used to immobilize Candida rugosa lipase on the γ-Fe2O3 nanoparticles. Published on Web 01/28/2003 1684 9 J. AM. CHEM. SOC. 2003, 125, 1684-1685 10.1021/ja021223n CCC: $25.00 © 2003 American Chemical Society