108 The 40 th ARA Proceedings Green Methods Used to Enhance Enzymes Mihaela D. Leonida Fairleigh Dickinson University, Teaneck, NJ, 07666, USA mleonida@fdu.edu Abstract: This project focuses on modifying properties of redox enzymes by using green chemistry. Working hypothesis: upon partially unfolding and subsequent refolding enzymes, new moieties can be embedded in their structure. These may confer additional/beneficial properties to the enzyme. Two methods were used to partially unfold the proteins: a) exposure to ionic liquids and b) application of high hydraulic pressure. The redox enzymes modified by exposure to an ionic liquid were lactate dehydrogenase (LDH), cholesterol oxidase (ChOx), and amine oxidase (AO). AO was also modified by transient exposure to high pressure. These are important oxidoreductases used in clinical laboratory, defense, sports medicine, the food industry. The kinetics of electron transfer is important when a redox enzyme is evaluated for an application because its redox centers are buried in insulating protein resulting in slow electron transfer. Since the rate of electron transfer in proteins decays exponentially with the distance donor-acceptor, we decreased the distances between redox centers within the enzyme by molecular alteration of its 3-D structure in the presence of modifiers. The choice of modifiers was tailored to the characteristics of each enzyme. Following the procedure, additional redox centers were entrapped within the protein structure thereby enhancing it. The enzymes were assayed before and after modification to assess the benefits of the procedures. All modified enzymes (ME) retained activity. The ME were tested as biosensing elements for analytical applications and performed well. The proposed methods are inexpensive, environmentally friendly, and enzyme friendly due to the species used as modifiers. 1. Introduction Oxidoreductases are needed for analytical applications (in biosensors) and for chiral synthesis of compounds with high enantiomeric purity for the pharmaceutical industry. However the rate of electron transfer in the reactions catalyzed by them is very slow when enzyme electrodes are used. A solution to the problem is the use of mediators to enhance the rate of electron transfer. One successful strategy to achieve this is covalently- binding redox-active centers (the mediators) to sites on the enzyme. This approach is known in the chemical literature as enzyme “wiring” and it typically results in an important loss in enzyme activity. Ionic liquids (IL) have gained interest lately due to their unique range of physical and chemical properties, notably their solvent capabilities, negligible vapor pressure and thermal stability [1]. Many commonly-used IL have excellent solvent properties for a wide range of organic, inorganic and organo-metallic compounds [2]. Their ability to dissolve proteins is of particular interest to bioorganic catalysis, IL replacing the traditional organic solvents in some applications [3]. Besides being environmentally unfriendly, organic solvents also affect adversely some suspended proteins [4]. In the present study we reversibly denatured redox enzymes by exposure to an ionic liquid 1-ethyl-3- methylimidazolium tetrafluoroborate (emim-BF 4 ). While the enzymes are partially unfolded with the redox centers exposed, they came in contact with species having electron transfer mediating capacity. Upon reversing the denaturation, small mediator molecules were trapped in the tertiary structure of the refolded enzymes which, consequently, become “wired” enzymes. (a) (b) Figure 1 (a) LDH from rabbit muscle; (b) ChOx from Brevibacterium sp.