Opening Lids: Modulation of Lipase Immobilization by Graphene

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Opening Lids: Modulation of Lipase Immobilization by Graphene Oxides Motilal MATHESH, Binquan Luan, Taiwo Akanbi, Jeffrey K. Weber, Jingquan Liu, Colin J. Barrow, Ruhong Zhou, and Wenrong Yang ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.6b00942 • Publication Date (Web): 09 Jun 2016 Downloaded from http://pubs.acs.org on June 12, 2016

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Opening Lids: Modulation of Lipase Immobilization by Graphene Oxides Motilal Mathesh⊥a, Binquan Luan⊥b, Taiwo Akanbia, Jeffrey K. Weberb, Jingquan Liuc, Colin J. Barrowa, Ruhong Zhoub,d* and Wenrong Yanga* a

Centre for Chemistry and Biotechnology, School of Life and Environmental Sciences, Deakin University, Geelong, Victoria-3217, Australia. b

IBM Thomas J. Watson Research Centre, Yorktown Heights, NY 10598, USA. c

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School of Chemistry, Qingdao University, Qingdao 266071, China.

Department of Chemistry, Columbia University, New York, NY 10027, USA.

ACS Paragon Plus Environment

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ABSTRACT

Lipases, which can be immobilized and reused for many reaction cycles, are important enzymes with many industrial applications. A key challenge in lipase immobilization for catalysis is to open the lipase lid and maintain it in an open conformation in order to expose its active site. Here we have designed “tailor-made” graphene-based nano-supports for effective lipase (QLM) immobilization through molecular engineering, which is in general a grand challenge to control biophysicochemical interactions at nano–bio interface. It was observed that increasing hydrophobic surface increased lipase activity due to opening of the helical lid present on lipase. The molecular mechanism of lid-opening revealed in molecular dynamics simulations highlights the role of hydrophobic interactions at the interface. We demonstrated that the open and active form of lipase can be achieved and tuned with an optimized activity through chemical reduction of graphene oxide. This research is a major step towards designing nanomaterials as a platform for enhancing enzyme immobilization/activity.

KEYWORDS: graphene oxides, lipase, hydrophobic, enzyme papers, simulation


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INTRODUCTION Lipases are a family of enzymes that, under natural conditions, hydrolyze carboxylic ester bonds in hydrophobic compounds such as triglycerides.1 Immobilized lipases are exploited in array of commercial biocatalysis applications, as fixed enzyme protocols have been demonstrated to yield high quality products at reduced processing costs with easy catalyst recovery. Despite their widespread use, however, the detailed molecular basis for the functionality of immobilized lipases has yet to be elucidated. Most lipases that rely on interfacial activation in vivo are thought to require the presence of a hydrophobic surface in order to adopt an open/active conformation.2 In general, lipases are known to possess a helical oligopeptide unit3 that, in its closed configuration, prohibits substrate access to the underlying active site. This helical motif is commonly referred to as lipase “lid”, which opens when placed at a hydrophobic interface to make the active site accessible.4 This is due to the recognition of hydrophobic supports by lipases and their adsorption through external region of the hydrophobic active centers, a phenomenon known as interfacial activation.5 This results in conformational changes in lipases resulting in opening up the lid.6 Various hydrophobic supports such as butyl-agarose and octydecyl-sepabeads have been used to immobilize lipases from Thermomyces lanuginose and Mucor Miehei

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respectively, which showed hyperactivation in comparison to free lipases.

Previous experimental results have also shown that adsorption on hydrophobic supports leads to open conformation while covalent immobilization leads to an equilibrium between open and closed forms which depends on reaction medium,9 hence demonstrating hydrophobic supports to be more suitable for immobilization. Increased interest in this kind of immobilization opened up an immense research field focusing on improved methodology that not only could permit hyperactivation and stabilization of enzymes but also release enzymes under drastic conditions.


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Various heterofunctional supports have been developed for this purpose like octyl-glyoxyl support which immobilizes the enzyme by interfacial activation and then covalently binds them to prevent enzyme release 10 for lipases from Alcaligenes sp.11 to improve its catalytic activity. In another instance, silica and sepharose supports with various functional groups were used to immobilize Geobacillus thermocatenulates lipase to study its stereoselective behavior.12 Although dramatic activation effects have been observed following adsorption onto hydrophobic supports and treatment with surfactants, it is not clear that such techniques lead to a maximal degree of lipase activity.2 Surfactants are important factor that could activate lipases by facilitating the lid opening. Several surfactants are available for lipase preparations like Span 85,13 Triton X-100

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and propylene glycol stearate,15 which mainly involve ion- pair formation

which could result in inactivated lipase

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or surfactant coating on lipases, which could contain

ester bonds, that will be degraded by lipase itself .4 In contrary, triton X-100 have been shown to stabilize the open form of lipases which could be immobilized onto aminated supports with the aid of glutaraldehyde, thus improving the catalytic activity.17 In this study, we have used lipase QLM from Alcaligenes sp. which is an extracellular enxyme with a molecular mass of 31 Kda, which are inhibited by cationic detegents.18 They have been used to produce important intermediates for pharmaceutical products.19 The interfacial activation of the lipase on hydrophobic supports has been studied previously, which showed improved catalytic activity.20 On the other hand, graphene oxide (GO) sheets which has been used here for immobilization consist of both aromatic nonpolar domains and oxygenated polar domains that endow them with the binary characteristics of a conventional block copolymer. GO edges are typically hydrophilic, possessing negatively charged carboxylate groups that promote binding to proteins like horse radish peroxidase via complementary electrostatic interactions.21 The more


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hydrophobic centers of GO nanosheets can also facilitate the dispersion interaction-driven binding of enzymes,22 eliminating the need for chemical modification of the protein prior to surface conjugation. GOs, which are known to act as surfactants 23, could potentially be used as a platform to immobilize lipases and to explore the mechanisms of surface-induced lipase lid opening.13 In fact, the mechanical strength, controllable surface chemistry, high specific surface area,24 and scalable manufacture of GO makes it ideal substrate for enzyme immobilization; to our knowledge, however, GO has yet to be utilized in such an application for lipase. Dispersed graphene sheets are known to interact with a wide range of active biomacromolecules.25 According to a recent study,26 GO can have a profound effect on the bioactivity of proteins suspended in solution. Conducted by Dravid and co-workers, a study involving α-Chymotypsin (ChT) demonstrated the capability of GO to inhibit enzymatic activity.27 By contrast, a subsequent study showed that PEG-functionalized GO can selectively improve trypsin-catalyzed proteolysis.26 Although the underlying molecular mechanisms behind these seemingly contradictory phenomena which needs to be clarified, such research indicates that dispersed GO can be utilized as a potential modulator of protein bioactivity. The diversity of macromolecular interactions between GO and proteins — involving various surface chemistries, structures, and conformational changes at nanoscale interfaces — highlights the scientific and practical importance of illuminating the statics and dynamics of GO-protein complexes at an atomic scale. One might then exploit the nature of such interactions to design new hybrid nanoparticle-biomolecule structures with myriad functions and novel folded architectures. As one pertinent example, the efficacy of enzyme-based heterogeneous catalysts has been demonstrated through the entrapment of lipases in hydrophobic sol-gel materials.28 While outstanding candidates for industrial applications, biomolecular catalysts integrated into


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synthetic scaffolds have thus far been limited by high fabrication costs and slow reaction kinetics resulting from reduced diffusion rates.29 Lipases, for one, are known to perform better in the presence of water,30 which acts as an effective lubricant during catalysis. Accordingly, a GO matrix interspersed with layers of water molecules (localized to the surface near oxidation sites) might be apt for use in lipase immobilization applications. GO’s planar structure could be further leveraged to fabricate many-layered enzymatic papers dense with highly active biocatalysts. In this article, we exploit this versatility of GO to design and build a new class of hybrid biocatalytic nanomaterials that facilitate molecular-level control of enzymatic activity. Through a novel design and regulation of GO reduction (inspired by an understanding of the lid opening mechanism derived from large scale molecular dynamics simulations), we illustrate how the activation and operation of immobilized lipase biocatalyts can be optimized.

RESULTS AND DISCUSSION

Lipase from the bacterium Alcaligenes sp. (QLM) is featured in our current study. The ability of the enzymes to hydrolyze pNPP and canola oil was investigated. After carrying out the lipase immobilization process (see SI Materials and Methods), the morphologies of bare GO sheets and GO sheets containing surface-bound QLM (GO+QLM) were visualized using AFM. In a manner consistent with previous studies,31 the height profile corresponding to bare GO sheets (Figure 1a) features a stable plateau near 1 nm (Figure 1b). Upon putative lipase immobilization (Figure 1c), a rougher surface topography emerges alongside a height profile plateau at 2-3 nm (Figure 1d), confirming that protein has been successfully deposited. These dramatic changes in the height profile likely correspond to the formation of a sandwich-like structure between two GO+QLM monolayers. Each GO+QLM sheet should be dotted with protein adsorbates on both sides,


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enabling the cohesion of multilayered complexes bridged by lipase molecules. Figure 1e shows a proposed molecular model for how QLM’s binding to single GO sheets proceeds.

Figure 1 Binding of lipase to graphenes. (a) AFM images of bare GO and (b) lipase-bound GO.

(c) Illustration of lipase adsorption onto GO obtained from molecular modeling. (d,e) Height profiles of representative cross sections taken from (a) and (b), respectively. The GO surface became rough upon lipase binding, and the corresponding height profile exhibits a doubling or tripling of sample thickness. (f) The far-UV CD spectra for lipase bound to different hydrophobic surfaces. A general decrease in ellipticity at 208 nm was observed, implying a decrease in α-helical content upon adsorption. Once lipase adsorption onto GO was confirmed, the effect of hydrophobicity on the immobilization process was investigated using chemically reduced GOs (CRGOs) with varying degrees of hydrophobicity/hydrophilicity, as synthesized by L-ascorbic acid (L-AA) reduction. GO and CRGO’s reduced for 1, 2, 3 and 4 hrs exhibited water contact angles of 28 º ± 4 º, 38 º ± 1 º, 46 º ± 1 º, 62 º ± 4 º and 70.6 º ± 2 º, respectively. Importantly, surface adsorption often leads


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to changes in the secondary structure of proteins.32 We thus employed CD spectroscopy to study the conformational changes brought about by QLM immobilization on different hydrophobic surfaces (Figure 1f), All CD spectra presented minima at 208 nm and maxima at 190 nm, indicating a general excess of α-helical content.33 As the surface hydrophobicity within the CRGO series increases, we observe a constant decrease in ellipticity at 208 nm — suggesting an associated decrease in α-helical content34 — after immobilization. This observation implies that α-helical structures are destabilized as oxygen-containing functional groups are systematically removed from the GO surface (Figure S2). Intriguingly, this spectral trend could arise from the binding of hydrophobic lipase lids to hydrophobic patches on GO scaffolds. It was previously reported that the immobilization of Burkholderia cepacia lipase (BCL) on a hydrophobic support (the microporous resin NKA) resulted in decrease in α-helical content and a concomitant opening of the BCL active site.35 It is thus conceivable that binding between QLM’s hydrophobic lid and hydrophobic regions on GO could disrupt the lid’s helical structure, a conformational change that might, in turn, significantly enhance QLM activity. MD studies (discussed in depth below) indeed support the notion that surface-induced lid opening becomes more prominent with increasing hydrophobicity. Notably, QLM immobilization onto highly reduced CRGO (treated for more than 4 hours) resulted in particularly drastic secondary structural changes in adsorbed enzymes (Figure S3). The corresponding CD spectrum’s minimum shifts to a value between 210 and 220 nm, and its maximum moves to around 195 nm; both such changes are characteristic of a transition to β sheet structures.36 Extreme surface hydrophobicity, thus, apparently leads to a crossover from α helices to β sheets in the immobilized enzymes, a transition which could lead to their deactivation (see further discussion below).


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To support the results obtained through CD spectroscopy, we performed FTIR studies to probe the systematic decrease in α-helical content observed with increasing GO reduction. Absorption peaks were measured in the 1900-1200 cm-1 region which comprises amide I (1700-1600 cm-1), amide II (1580-1510 cm-1) and amide III (1400-1200 cm-1) bands corresponding to respective C=O stretching, N-H bending, and C-N stretching modes.37 The amide I band is particularly sensitive to changes in protein secondary structure,38 yielding second derivative peaks at 1655 cm-1 and 1625 cm-1 signatory of α helices and β sheets, respectively.39 Past experiments have focused on these signatory bands to quantify the secondary structural characteristics of immobilized lipases from Candida rugosa37 and CALB.40 Spectral deconvolution of our FTIR data was performed using a 100% Lorentzian+Gaussian fit to derive α helix/ß sheet ratios. As shown in Table S1, the α helix/β sheet ratio decreased after QLM immobilization onto the various CRGOs, indicating a decrease in α helix content and an increase in β sheet content upon adsorption.


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Figure 2 Molecular dynamics simulation results. (a-b) Side and top views of the homology

model for the lipase QLM. The lid and catalytic domain are colored in red and grey, and the active site residue S89 is shown in a van der Waals sphere representation. (c-e) Simulation systems for the QLM on GO (c), GO/GR (d) and GR (e) nanosheets, respectively. Carbon, oxygen and hydrogen atoms in each nanosheet are shown in cyan, red, and white spheres, respectively. Water is rendered transparently and ions are not depicted. (f) Time-depenedent RMSDs for protein backbone atoms in different simulation systems (two independent runs for GO/GR, one each for GO and GR). The flexible N- (residues 1 to 11) and C-termini (residues 267 to 277) are not included in calculations. To better understand the molecular details of interactions between the lipase QLM and CRGOs, we carried out molecular dynamics (MD) simulations of QLM adsorbed onto various


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CRGO nanosheets. Figure 2a and 2b illustrate the side and top views of our atomistic QLM model. The enzyme’s active site (hallmarked by Ser89) is occluded by a lipase lid containing three helices (two long, and one short) that are joined by flexible connector regions. It is now well known that the oxidized groups on the graphene oxide are far from uniform -- they are randomly distributed but also strongly correlated with each other through the so-called "getting together effect." This effect is mainly due to the fact that once one of the carbon atoms in graphene is oxidized, its neighboring carbon atoms connected by π-π bond will become unstable, and hence are more prone to be oxidized. Similarly, when these neighboring carbon atoms are oxidized, they would further induce other neighboring carbon atoms to be oxidized.41 As a result, "chunks of hydrophobic (sp2-domain) regions and chunks of oxidized hydrophilic regions coexist on the GO",42 resulting in numerous GO domains, Graphene (GR) domains and GO/GR boundaries. Corresponding to these different CRGOs found in the experiment, we simulated three types of nanosheets: 1) a GO nanosheet that has the molecular formula C10O2H1 (highly oxidized, Figure 2c); 2) a GO/GR nanosheet wherein 50% of GO oxidation sites have been replaced by bare graphene (GR) atoms (Figure 2d); and 3) a GR nanosheet that contains no oxidization sites at all (Figure 2e). The dynamic processes of QLM adsorption onto these nanosheets were simulated independently, as shown in Figure 2 (c-e). Due to the complex nature of interactions between QLM and the GO/GR nanosheet, two separate simulations (labelled as GO/GR-1 and GO/GR-2) were performed in that case. Both of these simulation runs started from the same state wherein the QLM was initiated 5 Å above the interface between the GO and GR domains. Figure 2f shows the root mean square deviation (RMSD) of the QLM structure from its starting configuration. From analyses of four MD trajectories, the QLM was adsorbed onto each


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nanosheet within 10 ns of simulation time. After that, dynamic interactions between the QLM and nanosheets appear to affect the stability of the native protein structure. For the QLM bound to the GO sheet, time-dependent protein RMSDs saturate around 4 Å after tens of nanoseconds, indicating no significant changes in its secondary structure as well as in the entire conformation. However, after the QLM adsorbed onto either the GO/GR or GR nanosheet, protein RMSDs took over 100 ns to saturate and plateaued at values 7.7, 6.3 and 8.0 Å (for GO/GR-1, GO/GR-2, and GR nanosheets, respectively). These slow equilibration processes and considerable structural deviations suggest that non-trivial conformational changes occurred in QLM after adsorption. Trajectory analyses of MD simulations confirm that the QLM’s conformation can be substantially altered by interactions with graphene-based nanosheets. After QLM’s adsorption onto the GO nanosheet (Figure 3a), the lipase’s lid remained in an active site-obscuring position over the remainder of the simulation. This persistent obstruction of active site access perhaps explains the observed low activity of QLM immobilized on GO (see below in Figure 4). Owing to hydrogen bond-mediated hydrophilic interactions, the lipase molecule was completely immobile on the GO nanosheet surface (see movie S1). For the QLM that was initiated above the border between GO and GR domains, our two independent simulations demonstrated that the lipase could either be adsorbed in the border region (Figure 3b) or shift over to the bare GR domain prior to direct binding with the surface (Figure 3c). In GO/GR-1, the QLM was pinned to the border region by hydrophilic interactions with the GO domain (see movie S2). The lipase was much more mobile on the GR surface (in GO/GR-2) due to the relative translational invariance of hydrophobic interactions (see Movie S3 for an illustration of quasi-one-dimensional lipase diffusion on the GR domain). In some sense,


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QLM is not immobilized upon adsorption onto bare GO: the enzyme seems to bind to and diffuse across the two-dimensional graphene surface.

Figure 3 Conformational and energetic changes for the lipase QLM upon interaction with graphitic nanosheets. (a) The final conformation of QLM on the GO nanosheet. (b, c) The final conformations of QLM on the GO/GR-1 and GO/GR-2 nanosheets, respectively. (d) The final conformation of QLM on the GR nanosheet. (e) Angles between the lipase lid and support surface after QLM adsorption onto different nanosheets. (f) Time-dependent interaction energies between QLM and the graphitic nanosheets (yellow- GO, green-GO/GR 1, blue- GO/GR 2 and black- GR). (g) Proposed mechanism for the enhanced activity of QLM on a hydrophobic support: adsorption faciliates side-on substrate access to the QLM active site. The active site inside the QLM (transparent and cyan) is depicted as a red dot. In both GO/GR simulations, the lipase lid interacts with the GR domain via a collection of hydrophobic contacts. The trajectories indicate that such adsorption-mediated interactions


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outweigh associative interactions between the protein’s lid and catalytic domain, causing the lid to be pulled onto the GR surface. The consequent removal of the lid from the catalytic site, in principle, could afford substrates greater access to the lipase active site. Accordingly, increased enzymatic activity on more hydrophobic CRGOs could likely be attributed to strong interactions between the lipase lid and the GR (or sp2) domains on the GO surface. Over the course of our simulations, these hydrophobic surface interactions lead to helix-to-coil transitions within the lid domain (see Movie S3), an observation consistent with the reduced helical content seen in spectroscopic experiments (Figure 1f). It should be emphasized that our simulation time-scales (~ 300 ns) are not long enough, in general, to observe the formation of β-sheets. In the presence of the bare GR nanosheet, the QLM lid exhibits even more pronounced movement away from the enzyme’s catalytic domain (Figure 3d and Movie S4). After such a dramatic conformational change, the active site is completely open to substrate penetration. These data suggest that exceptionally strong hydrophobic interactions not only induce larger conformational rearrangements within the lid, but can also bias the lid’s position relative to the rest of the adsorbed enzyme. To quantify the opening of the lid’s “hinge” in relation to the protein body, as illustrated in Figure 3b, we define an angle θ among alpha carbon atoms in residues 151, 172 and 239 that are representative of lid, hinge, and catalytic domain positions, respectively. Prior to QLM adsorption, θ is fixed at approximately 11°; over the course of dynamics, θ reaches constant mean values of 20°, 38°, 46° and 65° on the GO, GO/GR-1, GO/GR-2 and GR nanosheets (Figure 3e). The hinge angle thus changes only slightly upon adsorption onto unreduced GO, indicating that hydrophilic interactions between the lipase and the GO nanosheet are ineffective in directing the lid-opening process. Progressively more dramatic hinge opening was observed in


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QLM bound to GO/GR and GR nanosheet surfaces, suggesting that enzymatic activity could be enhanced by surface interactions in both cases. Considering results derived from CD spectra, one might expect succeeding conformational changes on the bare graphene surface to interfere with activity enhancements. A system with improved catalytic functionality, thus, might be most easily realized at intermediate hinge angles (which should predominate at moderate levels of GO reduction). Once the structural changes were characterized, its effect on activity was studied with respect to the hydrolysis of canola oil and p-nitrophenyl palmitate (pNPP; Figure 4). In general, the activity of adsorbed QLM was observed to increase with rising surface hydrophobicity. It was also observed that QLM before and after immobilization in both cases had more specificity for pNPP than canola oil which is due to saturated and unsaturated fatty acid, respectively. The highest activity was observed for the CRGO surface exhibiting a water contact angle of 70.6 ± 2º. These increased activities can thus likely be attributed to interfacial activation, as observed for other lipases

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and indicated by both spectroscopic and simulation data. The activity

enhancement trend supports the veracity of the lid-opening mechanism suggested by MD simulations and corroborated by spectroscopically observed reductions in helicity upon QLM binding. An increase in available hydrophobic surface area on CRGOs likely leads to more pronounced interactions with the helical lid motif, facilitating hydrolysis by providing substrates with open access to the enzyme’s active site. There have been various beads prepared to study the effect of hydrophobicity on lipases. For example, four different hydrophobic magnetic porous microspheres were synthesized by copolymerisation of methacrylate and divinylbenzene. The highest activity observed was 1.8 times more than free lipases.44 Similarly, mesoporous silica foams has also been synthesized with varying level of hydrophobicity/hydrophillicity. The


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highest activity changes observed was 1.3 times of free lipase with respect to tributyrin.45 Despite these progress, there is a major drawback in terms of the synthesis of these particles with various levels of hydrophobicity, and the process is time consuming and requires special procedures. In our case, the activity is higher than these materials and the synthesis is facile and controllable.

Figure 4 Activity benchmarks measured in terms of canola oil (black) and pNPP (red) hydrolysis. Both benchmarks showed improved activity upon adsorption onto surfaces with higher hydrophobicity. The surface featuring the highest activity was rescaled to 100 % (N=3; error bars are SD). One might expect to find an optimal point up to which surface hydrophobicity has a positive effect on activity, and beyond which extreme hydrophobicity serves to disrupt catalysis. To place a bound on this optimization protocol, we adsorbed lipase molecules onto highly hydrophobic CRGO surfaces. As expected, we saw that excessive hydrophobicity acts to depress catalytic activity (Figure 4). The factors governing this deprecation in activity, one suspects, are twofold. First, although hydrophobicity increases catalytic activity (perhaps through the proposed


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adsorptive lid-opening mechanism), nearby hydrophilic surface regions could aid in attracting water necessary for hydrolysis.46 Secondly, the presence of broad hydrophobic surface regions might allow an adsorbed lipase lid to diffuse more freely across the landscape, motion ultimately detrimental to global protein structure and function.7 Measurements on the lipase TLL have demonstrated that its activity actually improves on hydrophilic surfaces as compared to purely hydrophobic surfaces; a large proportion of TLL molecules were found to be oriented with their active sites facing solution, suggesting an important role for locally wet surfaces in catalysis.46 The above evidence thus suggests that a lipase support material should contain a suitable degree of hydrophilicity to maximize catalytic activity. To better comprehend the decrease in enzymatic activity associated with high levels of hydrophobicity, we tracked the hydrophobic QLM residues most involved in interactions with the GR nanosheet in MD simulations (Figure S5). Though strong hydrophobic contacts prevent QLM desorption from the GR nanosheet surface, its lateral diffusion across the nanosheet is marked (see Figure S6). Thus, it is likely that adsorbed QLMs could encounter one another and aggregate on a bare GR surface, mutually occluding their active sites and thus reducing catalytic activity (Figure 2). Our GO/GR simulations suggest that such diffusion is effectively arrested by the presence of oxidation sites on the GR surface. As noted previously, exceptionally strong hydrophobic interactions with pristine graphene might also cause partial QLM unfolding, as seen in previous studies on the protein HP35

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and as supported by our spectroscopic data. Some

combination of these factors, perhaps, explains why enzymatic activity is disturbed once the hydrophobicity of GO surpasses a certain limit. Previous experiments have shown that hydrophobic mesoporous octyl silica can denature Cal B through overly strong hydrophobic


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adsorption;48 other enzymes (such as α-ChT) have exhibited depressed catalytic activity due to exceptionally strong interactions with their supports, as well.49 By analyzing the interaction energies, — including van der Waals and electrostatic energy components,— between the lipase and the various nanosheets simulated here, we find that equilibrated energies are more negative when the interactions are more hydrophobic (Figure 3f). For the QLM adsorbed onto the GR nanosheet, the total protein-GR interaction energy reached around -440 kcal/mol, more than four times that seen with the weakly interacting GO. Further rearrangement of auxiliary hydrophobic residues in the direction of the GR surface (a process that could lead to denaturation) may occur on experimental time scales. When hydrophobic interactions become more prominent, the absolute interaction energy between the QLM and the GO/GR nanosheet also becomes more favorable. These simulations highlight a new potential mechanism for the enhancement of enzymatic activity on hydrophobic support materials. One might hypothesize (see Figure 3g) that hydrophobic residues near the edge of the lid-body contact region would interact preferentially with the hydrophobic support medium, allowing the lid to swing open. However, the newly uncovered active site would still face the hydrophobic support in this configuration, likely resulting in suboptimal substrate binding. Thus, such a mechanism may not be applicable to QLM. Our simulation data suggest that the flexible lid deforms to maximize its interactions with the GR surface (Figure 3 b-d). Under such conditions, the exposed active site might be reconfigured to face bulk solution (Figure 3g), making it more accessible to substrates and thus facilitating higher catalytic activity. Encouraged by the above results, we took a further experimental step by fabricating a multilayered enzyme paper (Figure 5a) as a medium for high-density protein storage and


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biocatalysis. Cross-sectional imaging of this enzyme paper (conducted with SEM; Zeiss Supra 55 VP) reveals its multilayered structure (Figure 5b): planar rGO+QLM subunits (as shown in AFM) stack on top of one another to yield a three-dimensional rGO scaffold (Figure 5c). Raman spectroscopy confirms the presence of QLM within this rGO matrix. Two characteristic GO peaks were observed at around 1340 and 1580 cm-1, corresponding to D and G bands, respectively.50 The D band arises from defect-activated modes in sp2 hybridized domains on the rGO surface, while the G band is due to first-order scattering of the E2g phonon within graphene sheets.51 An increase in the ID/IG ratio from 0.958 to 1.113 was observed for rGO+QLM relative to protein-free rGO matrix (Figure 5d). This intensity shift is likely explained by QLM binding to hydrophobic regions on rGO, an interaction that would result in decreased peak intensity derived from sp2 domains and thus a reduced G band. A similar trend in the ID/IG ratio was observed previously in PCDO-GO binding studies.52


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Figure 5 Characterization of rGO+QLM paper. (a) A real-size image of the produced rGO+QLM paper. (b) SEM image (scale: 1µm) for a cross section of rGO+QLM paper, showcasing the multilayered structure of planar rGO+QLM motifs (Fig. 1) stacked on top of one another. (c) An illustration of multilayered structural motifs in rGO+QLM paper. (d) Raman spectroscopy of rGO paper and rGO+QLM paper. (e) FT-IR spectroscopy of rGO paper, free QLM, and rGO+QLM papers. (f) Second derivatives of FT-IR spectra corresponding to QLM and rGO+QLM papers. A decrease in α helix and an increase in β sheet content were observed for QLM immobilized on rGO paper. FT-IR was invoked to characterize the functional groups on rGO and to study the secondary structure of QLM before and after adsorption onto the GO surface (Figure 5e). rGO exhibited


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C=O vibrations at 1726 cm-1, O-H vibrations at 1620 cm-1, and C-O stretching at 1365 cm-1 53 in tandem with absorbance peaks at 1220 cm-1 and 1100 cm-1 due to epoxy

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and C-OH

vibrations.54 Curve fitting of spectra for QLM before and after immobilization demonstrated a decreased in α-helix/β-sheet ratio upon QLM interaction with rGO, a change that can be largely attributed to a reduction in α-helical content. However, second derivative analysis also showed an increase in β sheet structure upon enzyme-rGO binding (Figure 5f). This helix destabilization and sheet augmentation (also seen on single-layered rGO) might again be explained by the attachment of lipase lids to hydrophobic rGO domains (Figure 4). By all accounts, the mechanism for QLM’s adsorption onto multilayered rGO papers seems to be consistent with that seen for single rGO sheets.

Figure 6 Stability test for QLM immobilized in solution (yellow) and paper (blue) forms. Relatively stable behavior was observed in both forms for a period of 10 days; the paper form demonstrated the greater retention of activity after two months’ time. Here, basal activity at Day 0 is designated as 100 % activity. (N=3; error bars are SD). Inset: Absolute activities of rGO+QLM suspended in solution and within a rGO paper matrix, as measured by canola oil hydrolysis over 1 hour (N=3; error bars are SD).


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MD simulations suggest that the active-site opening in adsorbed QLM faces a plane normal to the rGO surface (Figure 4), a configuration that would allow bound enzymes to retain their activity after being stacked inside layers of rGO paper. To confirm the catalytic activity of these enzyme papers, the paper-induced hydrolysis of canola oil was measured and compared with that of rGO-immobilized enzymes suspended in solution (Figure 6 inset). An expected decrease in activity (~40%) was observed for the paper form, largely because of mass transfer limitations that do not appear in bulk solution. In particular, enzymes buried deep within the rGO matrix will naturally be less accessible to substrate, and therefore less active, than proteins adsorbed on the surface.55 The activity cost to using lipase paper for catalysis, however, promises to be compensated by considerable gains in long-term stability and recoverability. Stability tests demonstrate that both paper and solution forms of rGO+QLM remain highly functional over a period of 10 days, with solution-based enzymes retaining a surprising 95% of their initial activity (Figure 6). The rGO enzyme paper, however, was able to maintain 60% of its basal activity over a two-month time span; by contrast, enzymatic activity in solution declined to about 34% of its initial value over the same interval. These observations indicate that rGO enzyme paper may offer a viable solution for long-term lipase storage. For purposes of comparison, lipase from Candida rugosa immobilized on chitosan and encapsulated in inert sol-gel material showed 67% activity after 7 days and 50% activity after long-term storage;56 Rhizomucor meihie lipase adsorbed onto an ion exchange resin retained only 6% of its activity after 3 months of storage 57. Activity retention within rGO-lipase paper, thus, is very good in relation to previous efforts.

CONCLUSIONS


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Lipases are enzymes of exceptional importance to industry, hydrolyzing carboxylic ester bonds that appear in a myriad of food, detergent, pharmaceutical, and materials science applications. Here, we have demonstrated that selectively reduced graphene oxide can serve as an excellent lipase immobilization matrix that enhances catalytic activity in a scalable fashion. Careful control over the reduction of graphene oxide sheets yields a surface dotted with hydrophobic/hydrophilic interfaces. We find that such a landscape is primed for binding lipase molecules and encouraging lid-opening dynamics that do not interfere with enzyme mechanics. Experiments and simulations alike suggest that these patterned surfaces serve as apt modulators of lid-helix structure, adsorbing key residues in order to expose the lipase active site to substrates in solution. Simulation results indicate that hydrophobic surfaces can contort adsorbed enzymes into configurations in which the lipase active site is accessible from its side, enabling the design of stacked rGO-lipase scaffolds for storing, using, and recovering active biocatalysts. The multilayered enzyme paper we fabricated indeed exhibited sustained catalytic activity over a two-month period. Further study is certainly warranted to explore whether such GO-lipase complexes are practical for use on an industrial scale. Regardless, we here describe a marriage between biomolecular machines and inorganic nanomaterials that results in an extraordinary biocatalyst system, demonstrating that naturally evolved proteins can be remarkably functional in artificially engineered contexts. At all junctures, MD simulation data were key for guiding our design protocol. Intriguingly, we found that proteins presumed to be immobile on graphene were actually quite free to diffuse across the adsorbing surface (and, in principle, aggregate); this insight helped justify a patterning of hydrophobic and hydrophilic domains to improve catalytic activity. One might postulate that similar enzymes adsorbed onto hydrophobic support materials


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could benefit from the introduction of intermittent hydrophilic surface regions. In any case, similar syntheses of experimental and detailed simulation approaches should prove effective for guiding a myriad of biocatalysis applications in the future.

METHOD SECTION

SYNTHESIS OF GO AND CRGO

GO was synthesized using a modified Hummer’s Method

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with graphite flakes (Sigma-

Aldrich Pty. Ltd.) serving as the starting material. In a typical reaction, graphite flakes were initially oxidized with H2SO4 (Merck, 98% conc.) and ultrasonicated (VWR industries, water bath sonicator GRANXUBA3). The solution was dried at 70̊ C in a hot air oven and exfoliated with the help of H2SO4, KMnO4, and H2O2 (Sigma-Aldrich 30%). The resulting solution was ultrasonicated for 2 hours and washed with 1:10 HCl (Chem Supply, 32% w/w) and distilled water until its pH reached 7. CRGO was synthesized using a method from the literature,59 exploiting the reduction of GO via L-ascorbic acid (reagent grade, Sigma-Aldrich). As-prepared GO (10 ml, 1 mg/ml) was sonicated for 15 minutes and reduced using L-ascorbic acid (100 mg) under continuous stirring for different time spans. The reduction reaction was terminated by washing the solution three times with dH2O and subsequently centrifuging the mixture (Eppendorf centrifuge 5810R) at 11,000 rpm for 15 minutes. The resulting CRGO solution was adjusted to pH 7 using freshly prepared PBS buffer (pH 7.0) and used as such.

MOLECULAR DYNAMICS SIMULATIONS Simulation protocols and methods are provided in supporting information.


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AUTHOR INFORMATION Corresponding Author *Email for W.Y.: [email protected] and R.Z.: [email protected] Author Contributions W.Y. and R.Z. conceived and designed the research. M.M., T.A., J.L, and C.J.B. performed and analyzed all experiments. B.L. performed the molecular dynamics simulations and computational analyses. M.M., B.L., W.Y., and R.Z. wrote the manuscript with support from all authors. ⊥These

authors contributed equally.

Notes The authors declare no competing financial interests. ASSOCIATED CONTENT Supporting Information Materials and methods, activity tests, GO and CRGO characterization, MD simulation conditions. This material is available free of charge via the Internet at http://pubs.acs.org.

ACKNOWLEDGMENT We thank Bruce Berne and Seung-gu Kang for helpful discussions. RZ acknowledges the support from IBM Blue Gene Science Program.


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Table of Contents Graphic Table of Content


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Opening Lids: Modulation of Lipase Immobilization by Graphene

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