Amphiphilic polymer conetworks with defined nanostructure and tailored swelling behavior for exploring the activation of an entrapped lipase in organic solvents

Highlights

• Amphiphilic polymer conetworks activate inserted lipase in organic solvents.

• The activation is due to the nanophasic structure.

• If the organic phase swells separately relative activation is solvent independent.

Abstract

Amphiphilic polymer conetworks (APCNs) are nanomaterials that greatly activate entrapped enzymes in organic solvents. We have designed two novel APCNs with similar nanostructure, but different swelling behavior in toluene and n-heptane to explore the true origin of enzyme activation. They were realized by copolymerization of telechelic methacrylamide terminated poly(2-methyl oxazoline) (PMOx) with butyl acrylate (BuAc) and 2-ethylhexyl acrylate (EhAc), respectively. While the first APCN swells in toluene but not in n-heptane, the latter swells in both solvents. Lipase Cal B entrapped in the conetworks is most active at a composition that contains some 50 wt% PMOx in all cases. Further, the maximal activation of Cal B with respect to the suspended powder is some 20-fold independent on the solvent as long as the APCN is swellable.

Introduction

An amphiphilic polymer conetwork (APCN) is a multiphasic nanomaterial that combines orthogonal properties, which can be addressed separately via the nanostructures of two interconnected polymer phases [1], [2], [3]. This way, even materials can be created that swell in water and perfluorinated solvents [4] and show controlled ion conductivity [5]. Since amphiphilic polymer conetworks (APCN)s were first synthesized in 1988 [6], [7], their unique properties made them an object of research to understand and control their characteristics, which has resulted in high performance materials designed for numerous applications. Most popular is their use as soft contact lenses [8]. Recent research has shown the superior performance of APCNs as matrix for optical chemical [9] and biochemical [10] sensors, as drug delivery system [11], [12] as well as orthopedic tissue engineering [13] for medical research, as biodegradable materials [14], [15] and antifouling coatings [16], [17], [18]. Further, APCNs were found to be excellent membranes with pH- [19] and thermally [20], [21], [22] responsive swelling and porosity [23] as well as for chiral separation [24], [25], [26], [27] and selective diffusion [28]. The nanophases of APCNs can be loaded with enzymes [29]. Such bioactive conetworks have been shown to allow biomineralization from within the respective amphiphilic hydrogel [30].

One great feature of APCNs is their capability of activating enzymes in organic solvents [31]. Biocatalysis in organic media is an increasing field in the synthesis of fine chemicals [32], [33]. The advantages over enzymatic catalysis in aqueous reaction mixtures are better solubility of industrially interesting substrates, suppression of side reactions, and simple purification of the products [34], [35]. However, the activity of the enzymes in non-aqueous solvents is lowered by several orders of magnitude compared to water.

Many approaches to increase this activity concentrate on immobilization of the biocatalysts [34], [36], [37], [38], like adsorption on silica gel [39], small hydrogel particles [40], sepharose [41], or organic resins [42], entrapment in hydrogels or nanofibers [43] or direct crosslinking [44]. Others work on a further enhancement of entrapped catalysts due to radiofrequency treatment [45], additives [46], or simplified carrier removal by magnetic forces [47]. Alternatively, direct chemical modification of enzymes to get organo-soluble conjugates [48], [49] or protein engineering for organo-soluble enzyme mutants [50] have been described.

APCNs use their unique structure for entrapping biocatalysts in their hydrophilic nanophase, which prevents the enzyme from denaturing, and literally disperses the protein in the other organo-swellable nanophase. This hydrophobic phase swells and substrates diffuse to the interphase where biocatalysis takes place with high activity. This way APCNs can greatly activate entrapped enzymes in organic solvents [31], [51] and even in supercritical CO₂ [52]. APCNs with a chiral phase are not only activating entrapped lipases but also influence the enantioselectivity of the product formation [53].

The major drawback of previously prepared APCNs is the need to load the enzyme into the hydrophilic phase upon diffusion. This limits the loading capacity as well as the size of applicable enzymes greatly [4], [29]. These limitations were overcome by designing APCNs that can be prepared from a monomeric mixture with dissolved enzymes [54]. Interestingly, these networks are most active in a non-swelling solvent or a substrate mixture that swells the whole conetwork [55]. According to our concept, this should not be the case and might be explainable by the fact that the most active solvent system for the suspended enzyme is also the most active for the enzyme within the network, i.e., the solvent-specific activation is greater than the slowed diffusion of substrates through the non-swollen hydrophobic phase. In the present work, new APCNs were designed that can be prepared from prepolymer mixtures which are capable of dissolving enzymes and that swell the polymer phase orthogonal to the enzyme-containing hydrophilic phase. If the hydrophobic phase swells in n-heptane, we expect particular high enzyme activation due to faster substrate diffusion. These APCNs are used to explore the dependence of the activity of an entrapped enzyme in organic solvents on nanostructure and swelling behavior.