Enzyme immobilization on smart polymers: Catalysis on demand Giuseppe Cirillo a , Fiore Pasquale Nicoletta b , Manuela Curcio a , Umile Gianfranco Spizzirri a,⇑ , Nevio Picci a , Francesca Iemma a a Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, I-87036 Rende (CS), Italy b Department of Chemistry and Chemical Technologies, University of Calabria, I-87036 Rende (CS), Italy article info Article history: Received 19 March 2014 Received in revised form 27 May 2014 Accepted 5 July 2014 Available online 12 July 2014 Keywords: Thermo-responsive hydrogels On demand catalysis Enzyme immobilization Radical polymerization Pepsin abstract A new approach for the synthesis of hydrogel films with thermo-sensitive enzymatic activity is reported. Pepsin (PEP) was covalently immobilized on thermo-responsive hydrogels by radical polymerization in the presence of N-isopropylacrylamide and poly-(ethylene glycol) dimethacrylate 750, acting as functional monomer and crosslinking agent, respectively. Hydrogels showing lower critical solution temperatures between 32.9 and 36.1 °C were synthesized by UV-irradiation of reaction batches differing in the PEP/monomers ratio. The derivatization degree of the hydrogels was expressed as mg of PEP per gram of matrix and found to be in the range of 6 to 11% as assessed by Lowry method. Scanning electron microscopy analysis and water affinity evaluation allowed to highlight the porous morphology and thermo-responsivity of hydrogels as a function of temperature. Using bovine serum albumin as a substrate, kinetics parameters were determined by Lineweaver–Burk plots and the catalyst efficiency evaluated. The influence of temperature on enzyme activity, as well as the thermal stability and reusabil- ity of devices, were also investigated. Ó 2014 Elsevier Ltd. All rights reserved. 1. Introduction Enzymes are biocompatible and biodegradable catalysts from renewable resources, operating under mild conditions (room temperature, atmospheric pressure and physiological pH) in water, with high rates and selectivity [1–3]. Consequently, they are sustainable, environmentally friendly and cost-effective [4,5]. The application of enzymes in different industries (food, pharmaceuti- cal, chemical, textile) is continuously increasing, especially during the last two decades, in order to match the growing demand for green and sustainable chemicals manufacture [6–9]. Furthermore, immobilization of enzymes onto organic or inorganic polymer matrices has been developed to overcome some drawbacks associ- ated to their routine use, such as the lack of long-term stability and the difficulty in their recovery and reuse [10–15]. Immobilization allows the enhancement of the mechanical properties of enzymes and their stability to environmental changes [16,17]. In addition, the products can be easily purified without any contamination, and reused in continuous operational cycles [18,19]. Due to the high chemical versatility of the employed supports, a great variety of bioreactors for immobilization were fabricated [20,21]. Traditionally, three methods are used for enzyme immobi- lization, namely binding to a support (carrier), entrapment (or encapsulation), and cross-linking [16,17]. The binding to a polymer carrier can be reached by either physical (hydrophobic and van der Waals forces) or covalent interactions [22]. In the first case, the weakness of the involved interactions results in the formation of materials with low-term resistance to the hard reaction conditions of industrial processes, such as high ionic strengths and reactant/ product concentrations, leading to a desorption of the catalyst from supports [23]. Alternatively, a stronger linkage is obtained by the covalent coupling of the enzyme to a matrix, but the possi- bility to irreversibly deactivate the catalyst could occur, carrying out to un-effective materials [24]. The covalent bonds are generally formed through reactions involving the functional groups of unmodified enzyme side chains, such as lysine, cysteine, or aspartic and glutamic acid residues [16]. As an example, the exposed functional groups of these residues can react with supports bearing active esters, the most common are N- hydroxysuccinimide or epoxide-functionalized materials such as diglycidyl ethers. The most general method for the immobilization on aspartic and glutamic acid residues is their conversion into the corresponding active esters in situ with a carbodiimide coupling agent and an auxiliary nucleophile. As an alternative, the coupling http://dx.doi.org/10.1016/j.reactfunctpolym.2014.07.010 1381-5148/Ó 2014 Elsevier Ltd. All rights reserved. ⇑ Corresponding author. Address: Department of Pharmacy, Health and Nutri- tional Sciences, University of Calabria, Edificio Polifunzionale, I-87036 Rende (CS), Italy. Tel./fax: +39 0984493011. E-mail address: g.spizzirri@unical.it (U.G. Spizzirri). Reactive & Functional Polymers 83 (2014) 62–69 Contents lists available at ScienceDirect Reactive & Functional Polymers journal homepage: www.elsevier.com/locate/react