Enabling Multienzyme Biocatalysis Using Nanoporous Materials Bilal El-Zahab, Hongfei Jia, Ping Wang The University of Akron, Department of Chemical Engineering, Akron, Ohio 44325-3906; telephone: 330-972-2096; fax 330-972-5856; e-mail: wangp @uakron.edu Received 14 October 2003; accepted 9 March 2004 Published online 17 June 2004 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/bit.20131 Abstract: Multistep reactions catalyzed by a covalently immobilized enzyme-cofactor-enzyme system were achieved. Lactate dehydrogenase (LDH), glucose dehy- drogenase (GDH), and cofactor NADH were incorporated into two porous silica glass supports. One of the glass sup- ports had pores of 30 nm in diameter, while the other was of 100-nm pore size. Effective shuttling of the covalently bound NADH between LDH and GDH was achieved, such that regeneration cycles of NADH/NAD + were observed. The glass of 30-nm pore size afforded enzyme activities that were about twice those observed for the glass of 100-nm pore size, indicating the former provided better enzyme-cofactor integration. The effect of the size of spacers was also examined. The use of longer spacers in- creased the reaction rates by c 18 times as compared to those achieved with glutaraldehyde linkage. It appeared that the concave configuration of the nanopores played an important role in enabling the multistep reactions. The same multienzyme system immobilized on nonporous poly- styrene particles of 500-nm diameter was only c 2% active as the glass-supported system. It is believed that the nanoporous structure of the glass supports enhances the molecular interactions among the immobilized enzymes and cofactor, thus improving the catalytic efficiency of the system. B 2004 Wiley Periodicals, Inc. Keywords: multienzyme; biocatalysis; nanoporous; silica glass; cofactor regeneration; nanoparticles INTRODUCTION The exploration of multistep biotransformations, particu- larly those involving cofactor-dependent enzymes, consti- tutes an important part of today’s endeavors in ‘‘green’’ chemistry and chiral materials. Generally, cofactors can be regenerated via chemical, electrochemical, or enzymatic methods (Faber, 1995; Schaefer et al., 1986; Somers et al., 1997). Compared to other approaches, enzymatic regener- ation offers mild reactions and high efficiency that are preferred for most bioprocessing applications. To facilitate the reuse of the often considerably expensive cofactors and enzymes, efforts have been made to immobilize regener- ative multi-enzyme systems via both physical entrapment and covalent binding (Cosnier et al., 1999; Gambhir et al., 2001; Le and Means, 1998; Williams and Hupp, 1998). Such immobilized systems have been mostly useful in biosensing (Katz et al., 1998; Leca and Marty, 1997; Montagne and Marty, 1995; Ukeda et al., 1989), while it remains as a longstanding challenge to develop efficient multien- zyme catalysts for large-scale bioprocessing applications. Cofactor-dependent bioprocessing technologies developed so far primarily use free cofactors (Chang, 1987; Seelbach and Kragl, 1997), even though in some cases the cofactors were modified with polymers such as poly(ethylene glycol) for enlarged molecular size (Buckmann et al., 1981). In this work a triad catalytic system that consists of two enzymes and one cofactor was covalently incorporated into nano- porous glass supports, which have been proven especially effective in stabilizing enzymes (Wang et al., 2001). It is expected that the nanocavities of the glass will integrate the enzymes and cofactor into a molecular vicinity, and thus enable effective translocation of the cofactor between the active sites of the two coimmobilized enzymes. MATERIALS AND METHODS Materials Silica glasses of surface area of 50–100 m 2 /g were pur- chased from Silicycle (Quebec, QC, Canada). N-Acryloxy- succinimide (NAS) was obtained from Acros Organics (Morris Plains, NJ). Styrene and sodium hydroxide were obtained from EM Science (Gibbstown, NJ). Epichlorohy- drin (ECH), 3-aminopropyltrimethoxysilane (APTMS), and polyvinylpyrrolidone (PVP, Mw 29 kDa) were purchased from Aldrich (Milwaukee, WI). 2,2V-Azobis [2-methyl-N- (2-hydroxyethyl) propionamide] (VA-086) was provided as a gift from Wako Chemicals, Inc. (Richmond, VA). 2-Sulfoethyl methacrylate (2-SEM) was purchased from Monomer-Polymer & Dajac Labs, Inc. (Feasterville, PA). Ethanol (HPLC-grade), sodium phosphate, glutaraldehyde, B 2004 Wiley Periodicals, Inc. Correspondence to: Ping Wang Contract grant sponsor: National Science Foundation Contract grant number: BES-0117042