Tunable Enzymatic Activity and Enhanced Stability of Cellulase

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Tunable Enzymatic Activity and Enhanced Stability of Cellulase Immobilized in Biohybrid Nanogels Huan Peng, Kristin Rübsam, Felix Jakob, Ulrich Schwaneberg, and Andrij Pich Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b01119 • Publication Date (Web): 19 Sep 2016 Downloaded from http://pubs.acs.org on September 23, 2016

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Tunable Enzymatic Activity and Enhanced Stability of Cellulase Immobilized in Biohybrid Nanogels Huan Peng, a,c Kristin Rübsam,c Felix Jakob, c Ulrich Schwaneberg, b,c Andrij Picha,c*

a. Functional

and

Interactive

Polymers,

Institute

of

Technical

and

Macromolecular Chemistry, RWTH Aachen University, Aachen, Germany b. Institute for Biotechnology, RWTH Aachen University, Aachen, Germany c. DWI-Leibniz Institute for Interactive Materials e.V., Aachen, Germany

ABSTRACT: This paper reports a facile approach for encapsulation of enzymes in nanogels. Our approach is based on the use of reactive copolymers able to get conjugated with enzyme and build 3D colloidal networks or biohybrid nanogels. In a systematic study we address the question: how the chemical structure of nanogel network influences the biocatalytic activity of entrapped enzyme? The developed method allows precise control of the enzyme activity and improvement of enzyme resistance against harsh store conditions, chaotropic agents and organic solvents. The nanogels were constructed via direct chemical crosslinking of water-soluble reactive copolymers poly(N-vinylpyrrolidone-co-N-methacryloxysuccinimide) with proteins such as enhanced green fluorescent protein (EGFP) and cellulase in water-in-oil emulsion. The water-soluble reactive copolymers with controlled amount of reactive

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succinimide groups and narrow dispersity were synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization. Poly(ethylene glycol) bis(3-aminopropyl) and branched polyethylenimine were utilized as model crosslinkers to optimize synthesis of nanogels with different architectures in the preliminary experiments. Biofluorescent nanogels with different loading amount of EGFP and varying crosslinking densities were obtained. We demonstrate that the biocatalytic activity of cellulase-conjugated nanogels (CNG) can be elegantly tuned by control of their crosslinking degrees. Circular dichroism (CD) spectra demonstrated that the secondary structures of the immobilized cellulase were changed in the aspect of α-helix contents. The secondary structures of cellulase in highly crosslinked nanogels were strongly altered compared with loosely crosslinked nanogels. The fluorescence resonance energy transfer (FRET) based study further revealed that nanogels with lower crosslinking degree enable higher substrate transport rate, providing easier access to the active site of the enzyme. The biohybrid nanogels demonstrated significantly improved stability in preserving enzymatic activity compared with free cellulase. The functional biohybrid nanogels with tunable enzymatic activity and improved stability are promising candidates for applications in biocatalysis, biomass conversion or energy utilization fields. KEYWORDS: biohybrid, nanogels, enzyme, cellulase, activity, stability INTRODUCTION Hydrogels are crosslinked hydrophilic three dimensional polymeric networks which combine the properties of both solids and fluids.1 These soft materials are swollen


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with high water content, rendering significant small molecules to penetrate through the gel pore with fluid-like transport properties.2 Previous research reported that intelligent hydrogels can be responsive to a large variety of external stimuli from heat,3-5 light,6-8 pH,9-11 to enzyme,12,13 ionic strength14,15 and so on.16 These unique properties endow hydrogels intriguing potential applications in various fields such as drug delivery,17,18 catalysis,19,20 tissue engineering21,22 and sensors.23,24 Nanogels are colloidal polymer networks in tens to hundreds of nanometers in diameter, composed of chemically or physically crosslinked polymer chains swollen in a solvent (water). Although research on hydrogels is still prosperous at present, nanogels have already become an interdisciplinary research hotspot with enormous amount of publications on the preparations, properties and applications of nanogels.25-30 To date, among various techniques for nanogel formation, the most common route to synthesize chemically crosslinked nanogels is precipitation polymerization in aqueous solution.31 Particularly, it is facile to get homogenous nanogels with controllable properties via radical polymerization of water soluble vinyl monomers and cross-linkers. Nevertheless, the harsh conditions in this technique highly limited the application in bioactive nanogels preparation. In recent years, new routes were developed to break through limitations of traditional methods.32-35 Among these advancement, the particle replication in nonwetting templates (PRINT) method,36-39 as well as the microfluidics-assisted routes are widely reported techniques with promising applications.40-45 However, the special requirements in the aforementioned methods for chemical and mechanical design make the synthesis rather complicated


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and expensive. One of the strategies to increase the chemical diversity and complexity of nanogels for specific application is to design biopolymers as reactive building blocks for nanogel synthesis. The building blocks integrated with biological entities with tailorable properties can create a novel biohybrid nanogel system. Apart from the general properties of polymeric nanogels, such as high surface-to-volume ratio, tunable particle size and responsive to environment, the incorporated biological components offer the system additional functionalities. Although encapsulation of protein or enzyme in the polymeric nanogels also renders the colloids bioactivity,46 the drawback of instable locking property greatly weaken its application. Thus, the proteins need to be chemically or physically immobilized with the building blocks to keep a stable performance. Therefore, the chemically binding biohybrid nanogel system offers a stable platform combining the properties of both the building blocks and also the biological entities. Akiyoshi and coworkers reported pH-sensitive biohybrid nanogels via crosslinking vitamin B6 (pyridoxal) to hydrophilic polysaccharide, which may be utilized as nanocarriers in drug delivery system.47 Groll et al. reported nanogels with redox-responsive disulfide network by horseradish peroxidase (HRP) in the absence of hydrogen peroxide. The polymeric colloids demonstrated effective encapsulation of active β-galactosidase.48 Haag et al. formulated dendritic polyglycerol nanogels crosslinked with horseradish peroxidase in an inverse miniemulsion. This approach could encapsulate diverse catalytically active proteins like Candida antarctica lipase B.49 The design of the reactive building blocks is of critical importance to develop


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biohybrid

platforms

in

the

aspect

of

biocompatibility

and

architecture.

N-vinylpyrrolidone is widely used for biocompatible polymer synthesis50-52 while the facile conjugation between the succinimide group and free amino acid residuals was taken as an efficient tool to introduce biological componets.53-55. Maria S. et al. synthesized

poly((N-vinylpyrrolidone)-grafted-(aminopropyl)methacrylamide)

nanogels through e-beam irradiation of PVP aqueous solutions and studied the biomedical properties.56 Pollak A. et al. reported a convenient procedure for enzyme immobilization by functionalization poly(acrylamide-co-N-acryloxysuccinimide) with hexokinase.57 Although the conjugation between polypeptides and the succinimide groups undertook at mild conditions, the subsequent harsh process may disfavor the bioactivity of the nanogel. To ensure maximum protection of the protein, it should in a favorable environment once it was introduced into the system. Direct chemical-crosslinking between polypeptides and reactive polymers with predefined architecture, chemical composition and functionalities at very mild conditions in one step is a promising synthetic strategy. Our previous work thoroughly explored the RAFT polymerization of N-vinyl lactam monomers and N-methacryloxysuccinimide and reported the preliminary conjugation results between the reactive copolymer and cellulase.58 This systematic study addresses the question: how the chemical structure of nanogel network influences the biocatalytic activity and stability of entrapped enzyme? In present paper, water-soluble reactive polymers based on N-vinylpyrrolidone and N-methacryloxysuccinimide with controlled architectures were synthesized via RAFT


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polymerization. Biohybrid nanogels were synthesized systematically by crosslinking of reactive polymers with proteins (cellulase and EGFP) in water-in-oil emulsion at room temperature. In the preliminary experiments, polymeric nanogels crosslinked with

poly(ethylene

glycol)

bis(3-aminopropyl)

terminated

and

branched

polyethylenimine were prepared in different crosslinking densities. Enhanced green fluorescent protein (EGFP) and cellulase were utilized for biohybrid nanogels synthesis. The biocatalytic activity/stability of the cellulase-nanogels can be tunable via crosslinking density and the stability was enhanced compared with free cellulase. Circular dichroism spectroscopy was employed to investigate the change of the secondary structures of the biohybrid nanogels, and a fluorescence resonance energy transfer (FRET) based method was performed to study the influence of crosslinking density on substrate transport kinetics. The present work may open a new strategy to prepare bio-hybrid colloids with tunable catalytic activity and enhanced stability, demonstrating a tremendous potential in biotechnology applications.

EXPERIMENTAL SECTION Materials. N-vinylpyrrolidone (99%, Aldrich), N-methacryloxysuccinimide (98%, Aldrich), anisole (99.7%, anhydrous, Aldrich), methyl 2-bromopropionate (99%, Acros), potassium ethyl xanthogenate (>98%, Aldrich), poly(ethylene glycol) bis(3-aminopropyl) terminated (PEA, Mn~1500, Aldrich), branched polyethylenimine (PEI, Mn~600 by GPC, Aldrich), 4-methylumbelliferyl β-D-cellobioside (4-MUC, analytical grade, Aldrich), potassium phosphate buffer (PBS buffer, prepared by


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KH2PO4,

K2HPO4,

Aldrich),

3,3′-Dioctadecyloxacarbocyanine

sulfate

magnesium

perchlorate

(>99%,

(DiO,

VWR), Aldrich),

1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI, Aldrich), guanidinium chloride (GCl, >99%, Merck), urea (>99%, Merck), acetone (99.5%, Aldrich), methanol (99.8%, Aldrich), acetonitrile (99.9%, Aldrich), 1 2-propanediol (99.5%, Aldrich),

chloroform-d1(CDCl3, 99.8%, Deutero GmbH), dialysis tubes

(MWCO 12 kDa~14 kDa, Carl Roth, Germany; MWCO 50 kDa and 100 kDa, SPECTRUM® LABORATORIES, USA), and ethanol, dichloromethane, diethyl ether (99.9%,VWR) were used as received. 2, 2'-azobis(2-methylpropionitrile) (AIBN, 98%, Aldrich) was recrystallized in ethanol before use. Enhanced Green fluorescent protein (EGFP, with 19 solvent accessible lysine residues on the surface, M n~26 kDa) and cellulase (24 solvent accessible lysine residues on the surface, M n~69 kDa) were reengineered and purified according to previous report.59,60 Deionized water was used for all experiments. Synthesis

of

Reactive

poly(N-vinylpyrrolidone-co-N-methacryloxysuccinimide)

Copolymers by

RAFT

Polymerization. The water soluble reactive copolymer was synthesized by RAFT polymerization according to previous report.58 O-ethyl-S-(1-methoxycarbonyl)ethyl dithiocarbonate was synthesized first and used as chain transfer agent (Supporting Information). A typical procedure is described below. A stock solution in Schlenck flask containing N-vinylpyrrolidone (1 g, 9 mmol), AIBN (6 mg, 0.05 mmol), O-ethyl-S-(1-methoxycarbonyl)ethyl dithiocarbonate (0.021 g, 0.11 mmol) in 3 mL


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anisole was thoroughly deoxygenated by freeze-pump-thaw cycles. A vial charged with N-methacryloxysuccinimide (0.070 g, 0.38 mmol) in 1 mL anisole was sealed with rubber stopper and purged with dry nitrogen for 1h. Around 20 µL solution was taken out for

1

H NMR analysis before the reaction started at 70 ℃ . The

N-methacryloxysuccinimide solution was added into the flask progressively at 0.15 mL/h with the help of a syringe pump (Harvard Apparatus, Holliston, MA). The reaction proceeded for another 30 hours after all the solution was added to make sure high monomer conversion. The polymerization was stopped by immersion into liquid nitrogen. The copolymers were precipitated in excess amount of cold diethyl ether for 3 times and dried under vacuum for 48 h. Copolymers with 4%, 6%, 8% and 10% (initial molar feed ratio) N-methacryloxysuccinimide were named as CPn, (n=4, 6, 8, 10). All the polymers were synthesized and characterized in the same manner. More synthesis details as well as the NMR spectra of copolymers are presented in Supporting Information. Biohybrid Nanogels Synthesis. The synthesis route is illustrated in Scheme 1. Nanogels were synthesized via direct crosslinking reaction in a water-in-oil emulsion.58 A standard procedure is described as following. 6 mL toluene with 0.14 g span 80 was used as oil phase while the aqueous phase was 0.5 mL PBS buffer (0.02 M, pH 8.5) with 0.1 g CP10 (MSEC=7114 Da, reactive group%=11.9%, Supporting Information) and 1.5 mg PEA (molar ratio of succinimide/amine ~ 0.83). The aqueous solution was sonicated shortly to make the copolymer and cross-linker completely soluble. After the aqueous phase was added into the oil phase with a syringe, the


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solution was immediately ultrasonicated for 6 min using a Branson sonifier (200 W output, 30% duty cycle) under ice cooling. Then the emulsion was stirred at room temperature overnight. The nanogels were prewashed with centrifugation (12000 rpm, 16℃) and re-dispersion cycles to remove the hydrophobic surfactant span 80. Afterwards the nanogels were further purified by extensive dialysis (MWCO 12~14 kDa) in distilled water. The polymeric nanogels crosslinked with PEA and PEI are named as ANGn and INGn (n=4, 6, 8, 10), respectively. The synthesis of biohybrid nanogels was performed in a similar procedure. The oil phase was 6 mL toluene with 0.14 g span 80 was used as oil phase while 0.5 mL PBS buffer (0.02 M, pH 8.5) with 0.1 g CP10 (MSEC=7114 Da, reactive group%=11.9%) and 5 mg cellulase (or 2.5 mg EGFP) (molar ratio of succinimide/amine for cellulase~0.96, for EGFP, ~0.92) was used as aqueous phase. The aqueous solution was sonicated shortly to make the copolymer and cross-linker completely soluble before adding into the oil phase with a syringe. The solution was immediately ultrasonicated with a Branson sonifier (200 W output, 30% duty cycle) for 6 min under ice cooling. Then the emulsion was stirred at room temperature overnight and the nanogels were prewashed with centrifugation (12000 rpm, 16℃) and re-dispersion cycles. The nanogels were further purified by extensive dialysis in distilled water (under dark conditions for EGFP nanogels). In the case if EGFP and cellulase were used as crosslinking agents nanogels are named as ENGn and CNGn (n=4, 6, 8, and 10), respectively. The MWCO of dialysis tubes for purification of ENGn and CNGn were 50 kDa and 100 kDa, respectively.


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Scheme 1. Synthetic procedure to obtain biohybrid nanogels via crosslinking in W/O emulsion.

Fluorescence Spectroscopy Study. Fluorescence emission spectra were recorded on a Fluoromax-4 spectrofluorometer (Horiba Jobin Yvon GmbH, Germany). Excitation wavelength was set to 450 nm and the entrance slit and exit slit are 5 nm band pass. The spectra were analyzed with FluorEssence V3.5 software. Nuclear Magnetic Resonance Spectroscopy Study. High-resolution, 1H and

13

C

nuclear magnetic resonance (NMR) spectra were taken on a Bruker AV 400 spectrometer. Size-Exclusion Chromatography Study. Molecular weights (Mn, SEC and Mw, SEC)


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and molecular weight distributions (Đ, Mw/Mn) were determined by size-exclusion chromatography (SEC). SEC analyses were carried out with dimethylformamid (DMF) as eluent. Results were evaluated using the PSS WinGPC UniChrom software (Version 8.1.1). SEC with DMF (HPLC grade, VWR) as eluent was performed using an Agilent 1100 system equipped with a dual RI-/Visco detector (ETA-2020, WGE). The eluent contained 1 g·L-1 LiBr (≥99%, Sigma-Aldrich). The sample solvent contained traces of distilled water as internal standard. One pre-column (8×50 mm) and four GRAM gel columns (8×300 mm, Polymer Standards Service) were applied at a flow rate of 1.0 mL·min-1 at 40℃. The diameter of the gel particles measured 10 µm, the nominal pore widths were 30, 100, 1000 and 3000 Å. Calibration was achieved using narrow distributed polystyrene standards (Polymer Standards Service GmbH, Germany). Dynamic Light Scattering Study. DLS measurements were performed on an ALV setup consisting of a multiple s digital real-time ALV-7004 correlator, an ALV-SP8 goniometer, an ALV-SIPC photomultiplier, and a solid state laser (Koheras) with a wavelength of 473 nm at 20℃. The hydrodynamic radius and polydispersity index (PDI) were obtained by ALV-Correlator Software V3.0. Cryo-Field Emission Scanning Electron Microscopy Study. The cryo-scanning electron microscopy (Cryo-FESEM) images were taken with a HITACHI S-4800 instrument in a cryo-mode with secondary electron image resolution of 1.0–1.4 nm at voltages1–15 kV. Around 10 µL nanogel solution was put into the sample holder and immediately frozen with boiling liquid nitrogen. The frozen sample was then


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transferred to the high vacuum cryo-unit chamber and cut by a sharp knife to fracture the sample in the aim of getting images of the surface of the inner structure. The sample was sublimated for 3 min in order to remove humidity. The sample was then transferred to the observation chamber for measurement. Transmission Electron Microscopy Study. TEM was measured on Zeiss LibraTM 120 (Carl Zeiss, Oberkochen, Germany). The electron beam accelerating voltage was set at 120 kV. A drop of the sample was trickled on a piece of Formvar and carbon-coated copper grid. Before being placed into the TEM specimen holder, the copper grid was air dried under ambient conditions. Confocal Microscopy Study. The fluorescence images were recorded on a Leica SP8 confocal microscope (Leica, Germany). Bicinconinic Acid (BCA) Assay for Quantification of Protein in Nanogel. A calibration curve of defined concentrations of a protein solution was generated according to standard BCA assay procedure. The biohybrid nanogel solutions were measured in a multi-detection microplate reader (FLUOstar Omega, BMG Labtech, USA) with absorbance at 562 nm. The absorbance value of the nanogel was fit in the curve to get the protein concentration. Enzymatic Activity Assay. The activity assay was designed in the principal that 4-methylumbelliferyl-β-D-cellobioside (4-MUC) can be hydrolyzed into fluorescent 4-methylumbelliferone (4-MU) by cellulase in the nanogels.57 The experiments were performed in a fluorescence microtiter plate (MTP) reader Tecan Infinite M1000 Pro (Tecan Group Ltd, Switzerland), the excitation and emission wavelengths are 330 nm


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and 450 nm respectively, the gain is 140 manual. Circular Dichroism Spectroscopy Study. Circular dichroism (CD) spectra were recorded at room temperature in aqueous solution with an AVIV 62DS spectrometer (Aviv

Biomedical.

Inc.,

USA)

calibrated using

an aqueous

solution

of

(1S)-(+)-10-camphorsulforic acid (99%, Aldrich) at a concentration of 1.0 mg/mL. The samples contained a phosphate buffer at a concentration of 0.5 mg/mL. Data was collected in steps of 0.05 nm covering the spectral range between 250–190 nm. The bandwidth was 2 nm and the path length 0.1 mm. The raw data were smoothed by a least squares polynomial fit prior to evaluation. Substrate Transport Kinetics Simulation. The fluorophores encapsulated nanogels were synthesized according to following procedure.61 4 mg copolymer and 0.02 mg DiO or DiI fluorophore were dissolved in 200 µL of acetone and 2 mg PEI was added. After stirring for 10 min, 1 mL PBS buffer (0. 02 M, pH 7.2) was added and the mixed solution was stirred overnight at room temperature, open to the atmosphere allowing acetone to evaporate. Insoluble fluorophore was removed by filtration and excess amount of PEI and the released N-hydroxysuccinimide were removed from the nanogel solution by extensive dialysis (MWCO 12~14 kDa). 100 µL nanogel solution containing DiO mixed with equivalent nanogel solution containing DiI in a cuvette, and then 2 mL distilled water was added. The fluorescence intensity of the mixture solution was recorded periodically at 450 nm excitation wavelength. Stability Test. The enzymatic activity stability over time was carried out by storing the sample at room temperature and measuring the activity every 7 days. The stability


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against chaotropic agents was performed by mixing 60 µL PBS buffer, 10 µL chemical (0.08 M GCl or 0.1 M urea) and 10 µL sample in a microtiter plate (MTP). 20 µL 4-MUC was added immediately before the enzymatic activity assay started. The stability against organic solvents was performed by mixing 80 µL PBS buffer, 10 µL organic solvents (1 2-propanediol, methanol, acetonitrile or acetone) and 10 µL sample in a MTP, incubation for 30 mins at room temperature. Afterwards 20 µL of the mixture was discarded and 20 µL 4-MUC was added immediately before the enzymatic activity assay started. The tests were performed in triplicate.

RESULTS AND DISCUSSION Synthesis of Nanogels. To synthesize nanogels with different crosslinking densities, water-soluble reactive copolymers with varying succinimide ester groups and similar molecular weight (around 7700 Da~7860 Da, Supporting Information) were synthesized with RAFT polymerization. As observed from Figure S2, S3 and Table S1, the copolymers exhibit low dispersity and progressively increased amount of reactive groups from 3.97% to 11.9%. The much more reactive N-methacryloxysuccinimide was fed into the N-vinylpyrrolidone solution continuously to prepare copolymers with well-defined architecture. However, the contents of the succinimide ester groups are still higher than the feeding ratio due to the much higher reactivity ratio of

N-methacryloxysuccinimide compared with N-vinylpyrrolidone.58 Two types of cross-linkers with linear and branched structures were utilized to optimize the synthesis of nanogels. As observed from the cryo-FESEM images in Figure 1 and


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Figure S4, all the nanogels are with homogeneous spherical morphology and the nanogel sizes decrease with increased crosslinking degree. The highly crosslinked nanogels are more compact, leading to smaller particle size. The average radii of ANG nanogels obtained by image analysis of FESEM images are 153±7.8 nm, 121.5±10.4 nm, 76±9.5 nm and 67.5±8 nm for ANG4, ANG6, ANG8, ANG10 respectively, which are in good accordance with DLS results. The hydrodynamic radii of corresponding nanogels are 221±6.3 nm, 159±8.1 nm, 122±7.7 nm to 95±7.1 nm respectively (Figure S5). The larger size from DLS measurements may be due to partial volume loss during the freezing process. Similar trends could be observed from nanogels crosslinked with branched polyethylenimine. Interestingly, the nanogel sizes are larger than the corresponding ANG nanogels, as indicated by average radii of 181.5±10.2 nm, 136.5±12.9 nm, 108.5±10.5 nm and 82±8.2 nm derived from cryo-FESEM images (Figure S4) and hydrodynamic radius of 236±6.9 nm, 209±9.1 nm, 167±8.4 nm and 104±5.5 nm (Figure S5) for ING4, ING6, ING8 and ING10, respectively. This could be reasonable considering that the branched structure may cause increased steric hindrance, resulting in softer and larger colloids.


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Figure 1. Cryo-FESEM images of nanogels ANGn (A, n=4; B, n=6; C, n=8; D, n=10) (scale bar 500 nm).

The inserted scheme in Figure 2A illustrates the crosslinking reaction between the copolymers and the cross-linkers. The primary amine groups can react with the succinimide ester group in physiologic to slightly alkaline conditions (pH 7.2 to 9) to yield stable amide groups and release N-hydroxysuccinimide as byproduct, which was removed by the purification process. Figure 2A presents the FT-IR spectra of the copolymer (CP10), cross-linker PEA and the polymeric nanogel ANG10.The reaction ran overnight to get high The copolymer shows characteristic signals at 1776 cm-1 and 1806 cm-1 from –C=ON– of the succinimide groups, respectively while the peak at 1104 cm-1 is from –C-O– of the repeated ethylene glycol unit from the cross-linker. Compared with the two spectra, the FT-IR spectrum of ANG10 displays clear


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evidence of successful crosslinking reaction by observations of new peaks at 1524 cm-1 and 1104 cm-1 which are from the vibration of the newly formed –C=ONH– and –C-O– of the poly(ethylene glycol) chain respectively. Meanwhile the signals from – C=ON– of the succinimide group at 1806 cm-1, 1776 cm-1 disappear. It should be further noted that the peak at 1737 cm-1 of the copolymer is from the –C=O bond of the ester group. It shifts and overlaps with signals from the cyclic amide after the crosslinking reaction which transferred the ester group into amide group in mild conditions. Figure 2B demonstrates the FT-IR spectra of polymeric nanogels (ANG) with different crosslinking densities in a systematic manner. The peaks at 1661cm-1 of –C=O– from the lactam ring are normalized to have a better comparison of the peak intensities from the crosslinking network, which reveals the crosslinking density. As expected, the signals at 1524 cm-1 and 1104 cm-1 are progressively intensified from ANG4 to ANG10, indicating successful synthesis of polymeric nanogels with tunable crosslinking degree. Analogously, nanogels crosslinked with branched crosslinking agent PEI could be obtained with controlled crosslinking degrees, indicated by correspondingly intensified the peak signals at 1525 cm-1 in Figure S6. The free primary amino groups should demonstrate strong sharp double peaks at around 3250-3400 cm-1. However, as observed from the FTIR spectra, no such signals could be observed obviously, which indicates that most of the free amino groups were crosslinked during the crosslinking reaction.


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Figure 2. A) FT-IR spectra of copolymer CP10, crosslinking agent PEA and nanogel ANG10 (the right side images are enlarged areas where the arrows connect), the blue arrows point to the disappeared succinimide groups in the nanogels and the newly formed amide group respectively; the insert scheme represents the coupling reaction between the active ester group and the amine groups; B) FT-IR spectra of polymeric nanogels ANG4, ANG6. ANG8 and ANG10 (the right side image is enlarge part between 1750 and 800 cm-1; the peaks at 1661cm-1 are normalized), the red arrow points to the progressively intensified amide bond while the green arrow points to the –C-O– of the poly(ethylene glycol) chain.


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Biohybrid Nanogels. The biohybrid nanogels were prepared by direct coupling of reactive copolymers with EGFP or cellulase acting as cross-linkers. The reaction mechanism of the proteins and the reactive copolymers is similar to the reaction of the copolymers and the polymeric cross-linkers. EGFP and cellulase have 19 and 24 solvent accessible lysine residues respectively, the primary amino groups of which can react with the succinimide ester groups to form amide crosslinking network and release N-hydroxysuccinimide. The EGFP was utilized first to investigate the influence of synthetic environment on protein structure. As observed from the confocal microscopy images of ENG nanogels in Figure 3 and Figure S7, the nanogels are fluorescent and well dispersed, indicating the proteins were protected under the mild synthetic conditions. As expected, the hydrodynamic radius of nanogels decreases with increased crosslinking density as shown in Figure 3D (ENG4, 319±7.4 nm; ENG6, 270±8.7 nm; ENG8, 248±6.7 nm and ENG10, 226±6.9 nm). The loading amount of protein was determined by standard calibration method (Figure S8) and summarized in Table S2, suggesting high protein loading efficiency (all higher than 80%).


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Figure 3. A, B, C) Confocal microscopy images of ENG10 (A, bright field; B, fluorescent field; C, overlay image of A and B; the scale bars are 2.5 µm.); D) Particle size distribution of ENG nanogels measured by DLS.

Similarly, cryo-FESEM images in Figure 4 and DLS results in Figure S9 suggest that the cellulase-containing nanogels (CNG) possess similar good dispersion stability as the polymeric nanogels. The hydrodynamic radius decreases from 302±14.5 nm, 158±11.3 nm, 137±16.9 nm to 122±9 nm from CNG4 to CNG10 while the corresponding nanogel radii in frozen state decreases from 289.5±9.1 nm, 152.5±7.5 nm, 132±11.8 nm to 110.5±5.1 nm. These results indicate that the crosslinking density and colloid size of the biohybrid nanogels can be well controlled.


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Figure 4. Cryo-FESEM images of biohybrid nanogels CNG4 (A), CNG6 (B), CNG8 (C) and CNG10 (D). The scale bar is 1 µm.

FT-IR spectra of copolymer CP10, cellulase and nanogel CNG10 are compared in Figure 5A. The characteristic peaks of cellulase are 3354 cm-1, 3290 cm-1 and 1650 cm-1, 1580 cm-1 are the stretching and deformation vibrations of –NH2, at 1530 cm-1 is the vibration of –N-H– and at 1690 cm-1 for carboxyl group. It can be clearly observed that peaks at 1776 cm-1 and 1737 cm-1 from —C=ON— of the succinimide group disappeared while a new peak —C=ONH— at around 1524 cm-1 formed. Figure 5B displays systematic IR spectra of CNGs with normalized peaks at 1661 cm-1. The progressively increased peak intensities at 1524 cm-1 confirmed the corresponding increased crosslinking densities. Similarly, no obvious sharp double peaks were observed at 3250-3400 cm-1, indicating most of the enzyme participating


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Figure 5. A) FTIR spectra of copolymer CP10, cellulase and biohybrid nanogel CNG10 (the right image is enlarged part between 1500 and 1850 cm-1); the blue arrow points to the succinimide groups which disappeared in the CNG10 while the black arrow points to the newly formed amide groups in CNG10 after crosslinking. B) FTIR spectra of polymeric nanogels CNG4, CNG6. CNG8 and CNG10 (the right image is enlarged part between 1500 and 1640 cm-1; the peaks at 1661cm-1 are normalized); the progressively increased peak intensity at around 1530 cm-1 indicates increased crosslinking degree.

in the crosslinking reaction instead of grafting reaction. Standard BCA assay was performed to determine the loading amount of cellulase in


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the nanogels. The results are shown in Table S3, from which we can see that the loading efficiency are quite high, larger than 80%, indicating efficient proteins loading capacity of the biohybrid nanogels. It is reported that the cellulase is a promising catalyst for depolymerization of cellulose under mild conditions.59 In this paper a systematic study on the catalytic activities of nanogels with different crosslinking degrees was performed to have a better insight of the bioactivity of the enzymes entrapped in crosslinked colloids. Figure 6 demonstrates that the specific activities of the nanogels decrease largely compared with that of the free cellulase, which is 10.1 U/mg. Although nanogels CNG4 and CNG6 display rather high enzymatic catalytic activities, all the other crosslinked nanogels show much lower activities. It is supposed that the active sites of the proteins are in an unfolding or half-unfolding state in loosely crosslinked nanogels while they may be tightly arrested in highly crosslinked colloids. Another consideration is the accessibility of the active site of enzyme for the substrates, which may vary strongly with the crosslinking density and may contribute to the change of enzymatic activity. To address these concerns, a fluorescence resonance energy transfer (FRET) based study and circular dichroism characterization were performed to disclose the mechanism how the crosslinking density influences the enzymatic activity of the biohybrid colloids.


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Figure 6. A) Linearity of 4-methylumbelliferone (4-MU) fluorescence over 4-methylumbelliferyl β-D-cellobioside (4-MUC) conversion time; PBS buffer (0.2 M, pH 7.2) was used as control sample for measurements. B) Specific activities of biohybrid nanogels CNGn (n=4, 6, 8, and 10). One unit was defined as the amount of cellulase that catalyzes the conversion of 1 µmol of 4-MUC per minute.59 All values reported are the average of three measurements and deviations are calculated from the corresponding mean values.

We assume that the catalytic activity of the cellulase entrapped in biohybrid nanogels is mainly determined by the enzyme structure, and accessibility of the active site as well as diffusion kinetics of substrate molecules determined by the density of polymer network. A FRET based study was carried out to investigate the influence of crosslinking density on substrate transport rate across nanogels in aqueous solution. According to the principles of FRET, when the distance between the donor and acceptor fluorophores is within their Forster radius, the donor chromophore may transfer energy to the acceptor chromophore through nonradiative dipole–dipole


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coupling, making FRET especially sensitive to changes in small distance.62 The donor and acceptor fluorophores were independently sequestered in the same nanogels. When the both nanogel solutions mixed, the fluorophores would diffuse out into the bulk solution and further into the nearby nanogels. When the donor and acceptor fluorophores met inside the nanogels, the FRET phenomenon will be observed. In this way, the substrate transport dynamics can be monitored. Fluorescence spectra in Figure S11 show that the main absorption peaks of nanogel/DiO (donor) and nanogel/DiI (acceptor) systems are at 506 nm and 566 nm, respectively. From Figure 7A and Figure S11 one can observe that the peak intensities at 506 nm decrease with time while those at 566 nm increase accordingly. Figure 7B illustrate the substrate molecule transport kinetics. The slope of the fitting line is taken as the diffusion rate of the molecules. As demonstrated in the Figure 7B, the diffusion processes in the four selected nanogels are similar. Nearly constant diffusion rates are observed in the first 6 hours and then they gradually slow down to equilibrium. It can be clearly compared from the slops of the fitting lines in the first 6 hours that the transport rate in NG4/NG6 (0.02998 h-1, 0.02852 h-1) is much higher than those in NG8/NG10 (0.01439 h-1, 0.01089 h-1). This supports the hypothesis that substrates can diffuse into lower crosslinked nanogels at a higher speed, getting more effective access to the active sites of the cellulase in the nanogels.


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Figure 7. A) Fluorescence emission spectra of mixed nanogel based on NG4 encapsulated with DiI/DiO; B) Plots of FRET ratios in dependence of time of nanogels based on NG4, NG6, NG8 and NG10.

Circular dichroism spectroscopy was employed to investigate the state of the crosslinked enzyme. The cellulase contains 39.5% α-helix and 12.0% β-sheet secondary structures in its native form. Circular dichroism spectra in Figure 8 demonstrate a better insight of the protein structures in the biohybrid nanogels. The changes in the secondary structures of cellulase in the nanogels are detected by comparison of ellipticities at 222 nm, which are assigned to the α-helix contents in the protein. It is observed from Figure 8 that the amplitudes at 222 nm of biohybrid nanogels are attenuated compared with native cellulase, indicating varying states of the cellulase in nanogels with different crosslinking densities. The α-helix contents can be estimated from the mean residual ellipticity at 222 nm, θ222 with the following equation.63 α-helix%=-θ222/ 100 × (θ222/maxθ222)


(1)

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max

θ222 is the mean residual ellipticity value of one hundred percent helicity

polypeptide, reported as -31500 deg cm2 dmol-1 in previous literature.63 The calculated results of α-helix contents in native cellulase and biohybrid nanogels are demonstrated in Table 1. The α-helix contents decrease from 39.5% in native cellulase to 23.4% in CNG4, 16.7% in CNG6, 10.5% in CNG8 and to 7.9% in CNG10. The CNG4 nanogels encompass the highest α-helix content and accordingly demonstrate highest catalytic activity, which can only be initiated when the protein is in an unfolding state. The CD analysis confirms that secondary structures of protein in highly crosslinked nanogels were largely altered, making the chemically arrested enzyme in an almost-folding form. This state is further intensified in more highly crosslinked nanogels. Thus higher crosslinked nanogels demonstrate lower catalytic activity. Combined with the FRET study and CD results, it can be concluded that the catalytic activity of the biohybrid nanogel is mainly determined by the substrate transport rate across the nanogels and the secondary structures of the proteins. The lower crosslinked biohybrid nanogels possess larger substrate diffusion rates and the arrested enzyme is in more unfolding state. These two features can be tuned by the crosslinking density, providing a facile approach for controlling biocatalytic activity of the biohybrid nanogels.


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Figure 8. Circular dichroism spectra of cellulase and biohybrid nanogels CNG4, CNG6, CNG8 and CNG10.

Table 1. α-helix contents in biohybrid nanogels with different crosslinking densities -θ222 (deg cm2 dmol-1)

α-helix content (%)

Cellulase

11154.13

39.5

CNG4

8592.74

23.4

CNG6

7258.63

16.7

CNG8

5757.75

10.5

CNG10

5002.93

7.9

Stability Test. One of the important advantages for immobilizing cellulase is to enhance the enzymatic catalytic stability. The stability of the biohybrid nanogels was examined in different conditions. At first, the enzymatic activity stability was studied


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by storing these catalysts at room temperature (usually cellulase should be stored at 4℃). The specific activities of these materials were analyzed every 7 days. As demonstrated in Figure 9, the free cellulase loses more than 40% specific activity while all the biohybrid nanogels keep more than 77% residual activity after 3 weeks. It is worth to note that the biohybrid nanogels with higher crosslinking densities display better stability, keeping more than 80% residual activity. Interestingly, the activity of free cellulase decreased sharply at the first 7 days and became even lower in the following weeks while these of the biohybrid nanogels nearly reached equilibrium after sharp decrease in the first week. It is assumed that the chemical conjugation between the proteins and macromolecular network support the active sites of the protein, making the enzyme more flexible and robust even at room temperature. The covalently conjugations between cellulase and the polymeric nanogels supposedly decrease the hydrolytic activity while preserve the catalytic stability, which provides an advantageous strategy to immobilize cellulase with a stable efficiency and tunable activity.

Figure 9. A) Specific activities and B) residual activities of free cellulase, CNG4, CNG6, CNG8 and CNG10 over 21 days. Residual activity was defined as ratio of


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activity at 7/14/21 days and activity at 0 day.

Afterwards the stability test was performed by incubating the biohybrid nanogels with two kinds of widely used denaturants guanidinium chloride (GCl) and urea. These chaotropic agents can disrupt hydrogen bonding between water and water-protein and thereby may influence the protein structures and the associated unfolding processes. As demonstrated in Figure 10, the activity of free cellulase decreases sharply, with only 25.9% and 37.9% residual activity after incubation with the GCl and urea respectively. However, the biohybrid nanogels keep at least 43.1% and 51.5% residual activity in the same situation. The residual activity progressively increases to 61.7% and 69.9% in GCl and urea respectively when the crosslinking degrees increase. It could be ascribed to that the arrested enzymes in the crosslinked network were protected by the macromolecular chains, making it difficult for the chaotropic agents to access the proteins. This point could be supported by the above FRET experiments, which ascertained that molecules met more resistance to diffuse into highly crosslinked nanogels, demonstrating lower transport rates.


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Figure 10. A) Specific activities and B) residual activities of free cellulase, CNG4, CNG6, CNG8 and CNG10 incubated with guanidinium chloride (GCl), urea and PBS buffer (0.2 M, pH 7.2, control sample). Residual activity was defined as ratio of activity incubated with guanidinium chloride/urea and activity of control sample.

Considering the fact that many enzymatic catalysis processes can hardly be conducted in aqueous solutions due to the extremely low solubility of substrates as well as unfavorable shift of the reaction equilibrium in water, it would be interesting to study the catalytic stability of the biohybrid colloids in organic solvents. The organic solvents can disrupt the water shell of an enzyme molecule and may distort the hydrophobic interactions in the protein globule, leading to change of protein unfolding state. As displayed in Figure 11, the free cellulase loses more than 62.5% activity in acetone while the biohybrid nanogels keep at least more than 60% activity. It is reported that covalent conjugation hydrophilic –COOH groups on protein surface could help the enzyme to keep the hydration shell more tightly and resist the denaturation more efficiently caused by stripping of water molecules from organic solvents.64 The main components of the biohybrid nanogels are poly(vinylpyrrolidone)


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chains, which can tightly capture water molecules with hydrogen bonds and create a favorable aqueous microenvironment around the entrapped proteins, significantly hindering the access of the organic solvent to the protein surface. As observed in Figure 11, the biohybrid nanogels can protect the enzymatic activity of the proteins from different organic solvents. Furthermore, the residual activities are even higher in highly crosslinked nanogels. This indicates that the activity of the arrested enzyme can be well maintained in different organic solvents and the manipulated by the crosslinking degree.

Figure 11. A) Specific activities and B) residual activities of free cellulase, CNG4, CNG6, CNG8 and CNG10 incubated with organic solvents (1,2-propanediol, methanol, acetonitrile and acetone) and PBS buffer (0.2 M, pH 7.2, control sample). Residual activity was defined as ratio of activity incubated with organic solvents and activity of control sample.

The stability test well supports that the biohybrid nanogels possess robust enzymatic activity with wide tolerance with harsh store conditions, chaotropic agents and


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different organic solvents. These beneficial properties may help the biohybrid nanogels find applications in biomass utilization fields such as biofuels, plastic industry and fine chemicals.

CONCLUSION In

summary,

polymeric

nanogels

crosslinked

with

poly(ethylene

glycol)

bis(3-aminopropyl) terminated and branched polyethylenimine (ANG and ING respectivly) and biohybrid nanogels crosslinked with EGFP and cellulase (ENG and CNG respectively) were synthesized in water-in-oil emulsion. The nanogel size can be tunable via crosslinking density degree. The cryo-FESEM analysis demonstrated that the ANG and ING nanogels were well-defined spherical colloids, with diameters in the range of 135~306 and 164~363 nm, respectively. FT-IR spectra confirmed the successful crosslinking reaction by observing newly formed peaks at 1524 cm-1 and disappeared peaks at 1776 cm-1 and 1737 cm-1. The biohybrid nanogels conjugated with varying amount of enhanced green fluorescent protein were fluorescent and the particle sizes were tunable. The catalytic activities of cellulase-containing nanogels were adjustable by controlling the crosslinking densities. Nanogels with lower crosslinking degree demonstrated higher specific activities. Fluorescence resonance energy transfer (FRET) based study revealed that biohybrid nanogels with lower crosslinking degree enable higher substrate transport rate, making the substrate easier access the active site of the enzyme. Circular dichroism (CD) spectra indicated that the proteins were chemically arrested in the nanogel network with change of the


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secondary structure. The impact on the secondary structure of enzyme was enhanced with the increased crosslinking densities. Proteins entrapped in nanogels with lower crosslinking degrees were in a more unfolding state, resulting in higher enzymatic activities. The FRET study and CD results have disclosed the mechanism for controlling the catalytic activity of the biohybrid nanogels with crosslinking density. The stability test displayed that the chemically crosslinked enzyme nanogels were more stable than free cellulase in preserving enzymatic activity, exhibiting wide tolerance with harsh storage conditions, chaotropic agents and different organic solvents. Higher crosslinked nanogels demonstrated even better stability than lower crosslinked nanogels. The developed biohybrid nanogels displayed tremendous potential in various biotechnological applications like biomass utilization or biocatalysis. ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on ACS website. Characterizations of copolymers including 1H NMR, FT-IR, SEC data; Cryo-FESM image, DLS results, enzymatic activity, FRET experimental results, confocal images and TEM images of the nanogels. AURTHOR INFORMATION Corresponding Author *E-mail: [email protected] Author Contributions


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The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS H.P. thanks China Scholarship Council for financial support. A.P. thanks Volkswagen Foundation and Deutsche Forschungsgemeinschaft (DFG) with Collaborative Research Center SFB 985 „Functional Microgels and Microgel Systems“ for financial support.

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Tunable Enzymatic Activity and Enhanced Stability of Cellulase

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