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The use of enzymatic catalysis is a desirable approach to creating green and sustainable chemistry since this methodology helps to achieve high throughput under mild reaction conditions while featuring excellent chemo-, regio-, and stereo-selectivity (Koeller and Wong, 2001; Schmid et al., 2001). However, the limited operational stability of soluble enzymes due to denaturing and deactivation, as well as difficulties with their recovery and recycling, prevent their widespread use in industrial applications. This is why immobilization of soluble enzymes onto solid supports is an effective solution to those problems.
Although immobilization was first described almost 100 years ago (Nelson and Griffin, 1916), systematic investigations did not begin until the mid-1960s (Messing, 1975). Immobilization can provide for a number of benefits: (i) it often stabilizes the enzyme, (ii) it enables continuous use, (iii) the reaction is stopped after the reaction mixture leaves the reactor, and (iv) the product is not contaminated with the biocatalyst. On the other hand, immobilization may also have some disadvantages including decrease in activity due to distortion of the tertiary structure, blockage of the active site, and diffusional constrains of the mass transport (Garcia-Galan et al., 2011). The ideal immobilization technique should enable operation under mild reaction conditions. The best supports for the immobilization of free enzymes should (i) exhibit large surface areas, thus allowing immobilization of significant amounts of the enzymes, (ii) facilitate rapid mass transport of substrates and products, and (iii) provide excellent chemical and mechanical stability (Parthasarathy and Martin, 1994; Tischer and Wedekind, 1999).
A variety of techniques and solid supports have been described in the extensive literature including excellent recent reviews (Brady and Jordaan, 2009; Hernandez and Fernandez-Lafuente, 2011; Hwang and Gu, 2013; Mateo et al., 2007; Rodrigues et al., 2011). Typical immobilization techniques can be divided into two groups: physical methods such as adsorption, encapsulation, and entrapment, and chemical interactions including ion exchange and covalent attachment. In general, methods based on ion exchange are easy to implement. However, attachment of the enzymes may not be strong enough to prevent the loss of enzymes during the operation. In contrast, covalent binding is the most effective approach to averting detachment of the enzyme. However, it has been demonstrated that the chemical reaction of an enzyme with solid support leading to covalent bond may distort its three-dimensional conformation, causing partial denaturing, and a decrease in its activity (Blandino et al., 2000; Ozyilmaz and Gezer, 2010; Ozyilmaz and Yagiz, 2012). The activity of the immobilized enzymes also decreases during their operation. As a result, the solid catalyst must frequently be discarded and a new batch applied. Because of these drawbacks, new technologies are sought that will permit replacement of the enzyme in situ, that is, in the reactor, without losing the support.
This in situ replacement is currently possible by using ion exchangers and their metal complexes as a support (Ma et al., 2011; Wu et al., 2012). Most often, the bond between the support and enzyme is not sufficiently stable and use of certain substrate solutions or variations in pH lead to leak of the enzyme from the reactor. Therefore, development of novel support materials enabling strong but reversible binding of an enzyme is desirable.
Most of the supports for enzyme immobilization described in literature are porous particulates in which the mass transport of the substrates and products to and from the reaction site is achieved via diffusion through the stagnant liquid phase present in the pores. In contrast, porous polymer monoliths, which were developed in the early 1990s, are the newest contribution to the arsenal of supports (Svec and Fréchet, 1992). These monoliths are typically prepared from a mixture containing functional monomer, cross-linker, initiator, and pore forming solvents (porogens) using polymerization within the confines of a container where they are eventually used. Porous polymer monoliths contain large through-pores and exhibit high permeability to flow. These properties enable mass transport to the active site of the enzyme by fast convection rather than by slow diffusion since all the liquid phase must flow through the pores of the support. The advantages of monolithic supports have been demonstrated with several examples of immobilized enzymes used to process substrates dissolved in aqueous media (Bartolini et al., 2004; Josic and Buchacher, 2001; Krenkova et al., 2009; Logan et al., 2007; Mallik and Hage, 2006; Palms and Novotny, 2005; Pierre et al., 2006; Spross and Sinz, 2010; Sudheendran and Buchmeiser, 2010; Svec, 2006b; Svec and Fréchet, 1996; Xie et al., 1999). Recently, the application range of enzymes covalently immobilized on monolithic supports was extended to operations in both organic solvents and their biphasic mixtures with water (Kawakami et al., 2009; Urban et al., 2012). For example, Urban et al. prepared monoliths by polymerization of the reactive vinylazlactone. The azlactone functionalities readily reacted with amine groups of the porcine lipase to achieve covalent attachment of the enzyme onto the monolith. However, once the activity of the immobilized enzyme was decreased, the entire reactor had to be discarded.
Biodiesel is typically prepared by transesterification of triglycerides in fat with an alcohol in the presence of a chemical or biological catalyst (Al-Zuhair, 2007). The chemically catalyzed process can suffer from some drawbacks including slow reaction rate and necessity for high alcohol-to-oil ratios (Vasudevan and Briggs, 2008). In contrast, enzymatic transesterifications using commercially available lipases such as those from Thermomyces lanuginosus (e.g., Lipozyme TL IM), Pseudomonas antarctica (CALB, e.g., Novozym 435) or Rhizomucor miehei (e.g., RM IM) offer a number of advantages including (i) easy product separation, (ii) minimal requirements for wastewater treatment, (iii) easy glycerol recovery, and (iv) the absence of any side reactions (Jegannathan et al., 2008; Vyas et al., 2010). The high cost of lipases and contamination of the product with the enzyme are currently the major roadblocks to their wide application in large scale industrial transesterifications. Thus, immobilization of lipases on solid supports, and use of different techniques, was investigated to discover approaches that overcome these problems (Ozyilmaz and Gezer, 2010; Urban et al., 2012; Zhou et al., 2011). Garcia-Galan et al. (2011) described in a comprehensive review the potential of various enzyme immobilization strategies including covalent attachment and reversible immobilization focused on enhancement of the enzyme performance while using both porous supports and magnetic nanoparticles.
Previously, gold nanoparticles have been immobilized on monoliths and have been successfully used in the preparation of stationary phases for chromatography (Alwael et al., 2011; Cao et al., 2010; Connolly et al., 2010; Lv et al., 2012a, 2012c; Xu et al., 2010). Now, for the first time, we describe a universal approach to an easily prepared and highly efficient bioreactor using a support comprising a porous polymer monolith functionalized with gold nanoparticles. Commercially available porcine pancreatic lipase (PPL) was chosen as the model enzyme owing to its high stability and broad specificity for biotransformation of non-natural substrates (Bagi et al., 1997; Mendes et al., 2012).
Our bioreactor described in this paper consists of porous polymer monolithic supports with the pore surface coated with gold nanoparticles. The gold nanoparticles serve as an intermediate ligand on which the enzyme is immobilized (Scheme 1). In contrast to the conventional approaches, the enzyme is immobilized on gold nanoparticles via the reversible interaction of its thiol and amine functionalities with gold. The merits of this approach include: (i) high permeability and convective mass transport through the monolithic support rapidly delivering sufficient quantities of substrate to the enzyme; (ii) the increase in surface area contributed by the three dimensional gold nanoparticles attached to the otherwise “2-dimensional” pore surface enables immobilization of larger amounts of enzymes; (iii) the strong affinity of thiols and amines towards gold prevents leakage of the enzyme during operation; (iv) the interactions are achieved under very mild conditions; (v) the biocompatibility of gold nanoparticles is helpful in maintaining the native conformation and high activity of the biocatalyst; and (vi) the use of gold nanoparticles, as intermediate ligands at which the enzyme is non-covalently attached, facilitates regeneration of the activity of the bioreactor simply by removing the denatured enzyme, and subsequently recharging the bioreactor with a fresh enzyme.
Scheme 1. Preparation of monolith attached with gold nanoparticles, its immobilization with enzyme, and regeneration of the enzymatic bioreactor.
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