Graphene Oxide Nanosheets Shielding of Lipase Immobilized on

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Graphene oxide nanosheets shielding of lipase immobilized on magnetic composites for the improvement of enzyme stability Hongbo Suo, Lili Xu, Chao Xu, Xiang Qiu, Hongyue Chen, He Huang, and Yi Hu ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.8b06542 • Publication Date (Web): 25 Jan 2019 Downloaded from http://pubs.acs.org on January 28, 2019

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ACS Sustainable Chemistry & Engineering

Graphene oxide nanosheets shielding of lipase immobilized on magnetic composites for the improvement of enzyme stability Hongbo Suo1, 2, Lili Xu2, Chao Xu1, Xiang Qiu1, Hongyue Chen1, He Huang1,*, Yi Hu1,* (1State Key Laboratory of Materials-Oriented Chemical Engineering, School of Pharmaceutical Sciences, Nanjing Tech University, Nanjing 210009, China) (2College of Chemistry and Environmental Science, Qujing Normal University, Qujing 655011, China) *Corresponding authors He Huang, Professor Address: 5 New Mofan Road, School of Pharmaceutical Sciences, Nanjing Tech University, Nanjing 210009, China Tel. /Fax: +86-25-58139942. E-mail: [email protected] (H. Huang) Yi Hu, Professor Address: 5 New Mofan Road, School of Pharmaceutical Sciences, Nanjing Tech University, Nanjing 210009, China Tel. /Fax: +86-25-83172094 E-mail: [email protected] (Y. Hu)

ABSTRACT In this study, a novel enzyme immobilization method was developed to enhance the catalytic stability of enzyme. In this strategy, ionic liquid (IL) modified magnetic chitosan (MCS) composites were used as supports for lipase adsorption and graphene oxide (GO) was employed as shell coating for the first time. The modifier used was Imidazolium based IL with a side alkyl chain which was composed of 8 -CH2 and terminal hydroxyl group. The prepared supports IL-MCS, immobilized lipase PPL-ILMCS and GO/ PPL-IL-MCS were well characterized. The GO shielding lipase GO/

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PPL-IL-MCS maintained high activity (2468 U/g) which was 6.72-fold of free lipase. In addition, the pH and temperature effect on lipase activity were investigated. The thermal stability, denaturants stability, storage stibility and reusing stability were also studied. Compared to PPL-IL-MCS, the stabilities of GO/ PPL-IL-MCS were all enhanced while keeping high activity. For example, after 10 cycles of reuse, the residual activity of GO/ PPL-IL-MCS was 92.1% which was higher than that of 88.4% for PPLIL-MCS. Furthermore, the apparent Km of PPL-IL-MCS and GO/ PPL-IL-MCS was 5.7 mg/mL and 8.8 mg/mL respectively which were both lower than that of PPL-MCS (17.1 mg/mL). The Circular dichroism (CD) was used to analyze the secondary structure of lipase to explain the mechanism of stable enhancement of immobilized enzyme. This work demonstrated that GO was used as shell coating for the first time to improve the lipase stability. This immobilization method provides a reference for the immobilization of other kinds of enzymes. KEYWORDS: ionic liquids; graphene oxide; immobilized lipase; Magnetic chitosan nanocomposites; shield coating INTRODUCTION Biocatalysts exhibit broad application prospects in chemical, pharmaceutical and food industries due to their unique superiorities, such as high catalytic efficiency and selectivity, and mild reaction conditions1. However, the industrial application of enzymes is usually restricted by their low stability and difficult reusability2. Immobilization of enzymes can improve of the stability, selectivity or activity of enzymes and facilitate the recovery for reuse have received more and more attention3. Various supports for enzyme immobilization such as magnetic nanoparticles, metalorganic frameworks (MOFs)4, mesoporous silica5, graphene oxide (GO) and its derivatives6, biopolymers7 have been investigated. Proper host materials can improve the properties of enzymes. Magnetic nanoparticles possess superparamagnetism and high surface area can greatly improve the recovery and loading capacity. Compared to porous supports, magnetic nanoparticles have no diffusion limitations. Due to low toxicity and high stability, magnetite (Fe3O4) is the most commonly used magnetic particles8, 9. Xie and co-workers10 used graphene oxide


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encapsulated Fe3O4 composites to immobilize lipase from Candida rugosa (CRL) via the interfacial activation interaction for biodiesel production. The activity recovery and immobilization efficiency could be achieved 64.9% and 85.5%, respectively. Using the immobilized CRL, the biodiesel yield could reach 92.8% and the activity does not significantly loss after 5 cycles. Hosseini and co-workers11 reported cellulase was immobilized on cross-linked ionic liquid/epoxy type polymer entrapped Fe3O4 nanoparticles. The immobilized cellulase obtained enzyme capacity of 106.1 mg/g and retained 60% of its initial activity after 6 cycles. Many efforts have been done to explore new immobilization strategies to endow immobilized enzymes with high stability and specificity. For enzyme immobilization, physical adsorption is the most common and useful method which interact with enzyme via Van der Waals forces, ionic interactions, hydrogen bonding and hydrophobic interactions. This binding does not disrupt the active sites of enzyme and allows the enzyme to retain the conformational integrity12. However, shear force caused by liquid flow and stirring often lead to enzymes leakage. In order to prevent enzyme leakage, enzymes were confined and protected in MOFs or virus-like particles have been developed13. Coating a protected layer on the surface of pre-immobilized enzyme is another effective method. Various materials such as polydopamine (PDA)14, organosilicon12, polyacrylamide and protein biopolymer such as silk15 have been used as protective coating. Graphene oxide (GO) consists of a one-atom-thick planar sheet comprising an sp2bonded carbon structure and various oxygen functional groups which mostly in the form of hydroxyl, epoxy groups, carboxy, carbonyl, phenol, lactone, and quinone16. GO exhibits good chemical stability, biocompatibility, mechanical strength and large specific surface area and GO-based materials have attracted extensive attention16, 17. The above-mentioned advantages of GO-based materials endow it ideal support for enzyme immobilization18, 19. In the GO nanosheets, the aromatic nonpolar domains are hydrophobic and the oxygenated polar domains are hydrophilic. The hydrophobic regions can adsorb lipase through interfacial interaction which causing the lid opening and π-π stacking interaction. In addition, the hydrophilic regions consist of hydroxyl



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group, epoxides and carboxylate groups that can adsorb lipase via hydrogen bond and electrostatic interactions20. Furthermore, the GO nanosheets can provide a suitable microenvironment for biomolecule-immobilization while retaining their biological activities16. Surface modification of supports can affect the activity, enzyme capacity and stability of immobilized enzyme. Ionic liquids (ILs) have emerged as greener solvents due to their less-volatile, less flammable, low toxicity, and unique solubility for organic and inorganic materials. Biocatalytic reactions in ILs have been reported since 2000. ILs exhibited good biocompatibility and can stabilize and activate enzyme. In addition, use ILs as solvent, the lipases can exhibited good tolerance to extremely harsh conditions (high temperature and pressure)21. Also, ILs can provide a unique microenvironment around enzymes, in which a high enzyme activity was retained, resulting in excellent stability of the enzyme22, Novozyme

435

catalysis

23.

Itoh and co-workers reported

5-phenyl-1-penten-3-ol

((±)-1)

enantioselective

transesterification in [C4mim][BF4], after 10 cycles, the lipase retained excellent enantioselectivity in this IL as a solvent24. For industrial application of ILs in biocatalysis, the key is how to reduce the amount of ILs used while obtaining a certain benefit. Confined ILs in nanoporous hosts or used ILs to modify hosts are simple but versatile strategies to overcome this problem25. The enzymatic properties of Candida rugosa lipase and luciferase were improved respectively after immobilized on imidazole-based ionic liquids modified magnetic nanoparticles26, 27. Recently, we reported that porcine pancreas lipase (PPL) was successfully immobilized on ionic liquids (ILs) with different side chain length and different terminal groups modified magnetic chitosan nanoparticles28. Among the immobilized lipases, when the side alkyl chain of ILs composed of 8 -CH2 and with terminal hydroxyl group, the corresponding biocatalyst exhibited relatively enhanced stability and enzymatic properties. The novelty of the present study is using GO nanosheets as coating to shield the pre-immobilized lipase for anchoring the lipase and enhancing lipase stabilities (Scheme 1). Furthermore, as far as we know there is no literature reported on GO as protected layer on the surface has been reported. The GO coating


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can for further stabilized the immobilized lipase and enhanced the enzymatic properties through interactions with lipases. The prepared immobilized PPL demonstrated superiority in activity and stability compared to PPL on other matrixes29-31.

Scheme 1. Synthetic scheme of GO/PPL-IL-MCS MATERIALS AND METHODS Reagents and Materials Chitosan (CS, with 95% degree of deacetylation), 1, 1'-Carbonyldiimidazole (CDI) and 1-(3-Aminopropyl)-imidazole were purchased from Shanghai Aladdin Bio-Chem Technology Co., Ltd. FeCl2·4H2O and FeCl3·6H2O were purchased from Sinopharm Chemical Reagent Co. Ltd. 8-bromo-1-octanol was purchased from Energy Chemical (Shanghai). Porcine pancreatic lipase (PPL) was Sigma-Aldrich product. All other reagents used in this work were analytical grade. Synthesis of ILs Modified Magnetic Chitosan Nanoparticles Magnetic chitosan (MCS) nanoparticles were prepared using the co-precipitating method32 with minor modification. CS (0.25 g) was dissolved in 100 mL acetic acid solution (1%, v/v) and 2.0 g Fe3O4 nanoparticles were added. The resulting solution was stirred for 30 min and then 50 mL of 1 M NaOH solution were added to obtain Fe3O4@CS nanoparticles. The prepared nanoparticles were washed with deionized water until the pH reached 7.0 and then freeze-dried. The preparation of Fe3O4 was described in supporting information (SI). Dried (0.65 g) MCS nanoparticles and CDI (0.035 g) were added into 100 mL dichloromethane and the mixture were stirred at RT for 24 h. Then the CDI activated



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solids were separated and washed with ethanol for several times. The prepared solids and (0.125 g) 1-(3-Aminopropyl)-imidazole were added into 50 mL DMSO and reacted for 24 h at RT. The obtained solids (AIm-MCS) were washed with ethanol for several times. Subsequently, (0.5 g) MCS and (0.21 g) 8-bromo-1-octanol were dispersed in acetonitrile, and were allowed to react at 80 C for 24 h. Then, the products were collected and washed with ether for several times. Then, the synthesized solids and KPF6 (0.92 g) were dispersed in deionized water (100 mL) to complete ionic exchange. The obtained solids were IL modified MCS nanoparticles (IL-MCS). The XRD pattern of supports was shown in Figure S1. Preparation of Immobilized Lipase The as-prepared IL-MCS (0.5 g) was added in 50 mL PPL solution (1.37 mg/ml, pH=7 PBS solution), and the mixture was incubated at 30 C for 5 h under shaking condition (150 rpm). After that, the immobilized PPL (PPL-IL-MCS) was separated by an external magnet, and washed with PBS buffer for three times. The immobilized lipase was suspended in 50 mL PBS buffer (0.025M, pH=7.0) and stored at 4 C. The optimization of immobilization conditions were shown in Figure S2. Graphene oxide (GO, 0.03 g) was added to 50 mL PBS buffer (0.025M, pH=7.0) under lengthily ultrasonic treatment for 3 h. Subsequently, the as-prepared 50 mL immobilized lipase suspension was added in the 50 mL GO colloid solution under slightly stirring at RT. After reacted for 30 min, PPL-IL-MCS was shielded in GO. Then, the GO shielded lipase (GO/PPL-IL-MCS) was separated magnetically and washed with PBS buffer for three times and then freeze-dried. During the process, Bradford method was used to measure protein concentration33. The amount of lipase immobilized onto supports was calculated by the following equation (Eq. 1) Enzyme loading 

CiVi - CsVs  CwVw m

Eq. 1

Where Ci, Cs and Cw are the lipase concentrations of initial enzyme solution, supernatant and buffer washings, respectively. Vi, Vs and Vw are the volumes of initial enzyme solution, supernatant and buffer washings, respectively. And m is the mass of added support.


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Enzymatic Activity Assay The activity of free and immobilized lipase were measured using triacetin as substrate in PBS buffer with a certain pH. Briefly, 0.1 g immobilized lipase was added in 20 mL homogenized triacetin buffer solution (3.4% w/v) and the mixture reacted at 35-65 C with shaking (150 rpm) for 10 min. After completing the reaction, the immobilized lipase was separated by an external magnet, and the solution was titrated with 0.05 M NaOH solution. According to the consumed volume of NaOH solution, the activity of free and immobilized lipase were calculated. One unit of lipase activity was defined as the amount of enzyme required to release 1 μmol of acetic acid per minute. All experiments were performed for three times. Optimization of Enzymatic Reaction Conditions The optimum reaction pH and temperature of the immobilized lipase were investigated using the activity assay procedure mentioned above. The pH of the reaction solution was kept at 7.0 when the temperature ranging from 35-65 C. When adjusting the pH by PBS buffer (0.025 M) from 5.5 to 8.5, the reaction temperature was kept constant at 50 C. Operational Stability of Immobilized Lipase The thermal stability was investigated by measuring the residual activity of enzyme when immobilized lipase was incubated in PBS buffer under optimum pH at 50 C for 1 to 6 h, respectively. The stability against chemical denaturant was tested by incubating samples in urea solution with concentration ranging from 1M to 5M for 2h. Then the residual activity was measured under optimum conditions, respectively. To assay the reusability of immobilized lipase, the hydrolysis reaction was performed for 10 cycles at 50 C with pH 8.0. After one cycle, the immobilized lipase was collected magnetically and washed for three time with PBS buffer (pH 8.0). Then the collected immobilized lipase was re-suspended in a fresh reaction solution for the next cycle. The storage stability was measured by storing the immobilized lipase at 4 C for a period of time. The residual activity was tested every 5 days and repeated for 7 times.



The measurement of residual activity was carried out under optimum conditions. The relative activity of each sample referred to the radio of the residual activity to the original activity. All the stability experiments were studied under optimum reaction conditions and repeated at least three times. Kinetic Measurements To evaluate Michelis-Menten constant (Km) and the maximum reaction velocity (Vmax) of free and immobilized lipase, the iniatial reaction rates were measured with a substrate concentration ranging (9-30 mg mL-1) for 3 min reaction under respective optium temperature and pH. The apparent Km and Vmax could be calculated from a LineweaverBurk plot accordingly. Characterizations Fourier transform infrared (FTIR) analysis was performed on a Nicolet Nexus 670 spectrometer (Thermo, USA) in the range of 400-4000 cm-1. Scanning electron microscopy (SEM) was recorded on a Hitachi S4800 (Japan). Thermogravimetric analysis (TGA) was performed on a NETZSH STA 409 PC (Germany) with a heating rate of 10 C min-1 from 30- 800 C under N2 atmosphere. The magnetic properties of the samples were investigated using a vibrating sample magnetometer (VSM, Quantum Design, USA) at room temperature (RT) with a magnetic field ranging from 30 to -30 KOe. Circular dichroism (CD) (190−250 nm) was recorded on a JASCO J1500 (Japan) spectrometer at RT. The X-ray diffraction (XRD) spectra was conducted on a Bruker D8-Advance diffractometer equipped with a Cu/Ka radiation at a scanning rate of 4-8° min-1 at RT. The hydrophobicity test of supports was evaluated by a contact angle instrument (DSA100, Germany) with a water drop (5μL) at RT. The values of contact angle were determined using sessile drop mode. RESULTS AND DISCUSSION Synthesis and Characterization of Supports The magnetic properties of supports were investigated by VSM at room temperature and the magnetization curves were shown in Figure S3. All the samples exhibited reversible hysteresis behavior and zero coercivity, which confirmed their super-


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paramagnetic behavior at room temperature34. The saturation magnetization (Ms) of Fe3O4 was 68.4 emu g-1, and declined after Fe3O4 coated with CS, modified with IL and incorporated with PPL and GO. The Ms of prepared biocatalysts PPL-IL-CS-Fe3O4 and GO-PPL-IL-CS-Fe3O4 were 55.5 and 51.6 emu g-1, respectively. They could be separated easily from the reaction system using a magnet, which improved the reusability of the immobilized lipase.

Figure 1. FTIR spectrum of (a) CS, (b) IL-CS, (c) Fe3O4, (d) IL-MCS, (e) PPL-ILMCS and (f) GO/PPL-IL-MCS. The FTIR spectrum of different samples were showed in Figure 1. The characteristic adsorption peaks of chitosan at 3436 cm-1 was ascribable to O-H and N-H stretching vibration, 2869 cm-1 was assigned C-H stretching vibration, 1596 cm-1 N-H corresponded to bending vibration and 1083 was attributed to C-O-C stretching vibration28 (Figure 1a). There was a new adsorption peak appeared at 1525 cm-1 for ILCS (Figure 1b), this signal would be attributed to carboxyl in urea group35. The peak at 580 cm-1 was the characteristic adsorption of Fe3O4. After immobilization of lipase, the spectrum of PPL-IL-CS- Fe3O4 and GO- PPL-IL-CS- Fe3O4 showed new adsorption bands at 1533 cm-1 which belonged to the deformation vibration of NH2 groups from PPL14, 30. The results confirmed that IL was successfully grafted to CS and PPL was immobilized on the supports.



Figure 2. 1H-NMR spectra of (a) IL-CS and (b) CS. The 1H-NMR spectra of CS and IL-CS were shown in Figure 2. The characteristic proton signals for chitosan appeared at 1.80 and 1.90 ppm for H1 and H7, 2.99 ppm for H2, 3.54-3.74 ppm for H2 - H6 in Figure 2b which were consistent with the literature36. As shown in Figure 2a, after modified with IL, IL-CS exhibited all characteristic peaks of CS. In addition, the signals for IL-CS were observed at 1.01 ppm were assigned to the methylene of side chain of IL (H16-H21), multiple signals at 1.83-1.89 ppm were attributed to H9, H10, H15, multiple signals at 4.19-4.24 were related to H8, H11. Furthermore, signals at 8.62, 7.39, and 7.32 ppm were assigned to H12, H13, and H14 of imidazolium ring28. These results indicated that chitosan was modified by IL successfully.

Figure 3. TGA of (a) Fe3O4, (b) MCS, (c) IL- MCS, (d) PPL- IL- MCS and (e) GO/PPL-IL- MCS.


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Figure 3 showed the TG analysis of different samples. As shown in Fig. 3a, Fe3O4 was thermal stable in the temperature range, only lost 7.9% of its weight. After coating CS, the weight loss of MCS was 12.6%, indicating the chitosan amount in MCS was about 4.7%. IL-MCS and MCS exibited similar weight loss trend before 300 C, from 300 C onwards, the imidazole ring of IL-MCS began to decompose37 and the weight loss reached 14.7% at temperature 800C as shown in Fig. 3c. Compared to IL-MCS, the supported lipase sample (PPL-IL-MCS) showed sharp decrease in weight from 250 C onward, due to the decomposition of lipase26 and lost of 20.5% of its weight in the testing temperature range. After shielding with GO, the weiht loss of GO/PPL-IL-MCS reached 24.8%. TGA results improved CS was modified with IL, lipase was immobilized on ILmodified nanoparticles and shielded with GO.

Figure 4. SEM images of (a, b) PPL- IL-MCS and (c, d) GO/PPL-IL-MCS. The morphologies and microstructures of immobilized lipase were given in Figure 4. It can be seen from Figure 4 (a, b), the supports were spherical or ellipsoidal, which can provide large specific area for enzyme immobilization. GO exhibited crumpled and rippled sheet structure38 (Figure 4c), which can provide large specific area and favorable microenvironment for the immobilized PPL. The secondary structure of immobilized lipase may not be disrupted due to the excellent biocompatibility of GO19.



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The GO nanosheet can anchor PPL-IL-MCS through π-π stacking , Van der Waals forces, and hydrogen bonding interactions39. The GO nanosheets coating can prevent the leaching of lipase that can stabilize the operation. Moreover, after adsorbed on the surface of GO, the immobilized lipase can dispersed well on the nanosheets. (Figure 4d). Enzymatic Properties of Immobilized PPL Immobilization of Lipase and Activity Assay Table 1. Results of immobilization of PPL Samples

Immobilization

Lipase content

Specific activity

yield (%)

(mg/g)

(U/g)

PPL-MCS

79.9 ± 2.1

110 ± 2.4

768 ± 8.5

PPL-IL-MCS

91.5 ± 2.8

126 ± 3.6

2615 ± 7.9

/

115 ± 3.3

2468 ± 7.6

GO/PPL-IL-MCS

The activity of free PPL: 367 U/g at 45 °C, pH 7.0.

The immobilization results of lipase were shown in Table 1. Compared with PPLMCS, lipase immobilized on ILs modified MCS presented higher enzyme capacity and specific activity. The result may be attributed to the introduction of ILs. Our previous work demonstrated that with the increase of carbon chain of IL, the loading mount and activity of immobilized lipase also increased. When the carbon chain increased to a certain extent, the activity of immobilized lipase was decreased28, 37. The side chain of ILs contained 8 of –CH2 that enhanced the hydrophobicity of support (Figure S4). Surface hydrophobicity of supports had positive effect on lipase activity. As reported40, 41,

the hydrophobic supports could open the lipase lid through interfacial activation that

made the active center accessible. Also, lipase could be adsorbed on the surface of supports through interfacial activation. The specific activity of PPL-IL-MCS was 2615 U/g, 7.1 folds to that of free PPL and 3.4 folds to that of PPL-MCS. In order to stabilize the open form of immobilized lipase, we used GO nanosheets as a coating to shield the immobilized lipase. After coated with GO, the specific activity of GO/PPL-IL-MCS was 2468 U/g, slightly lower than that of PPL-IL-MCS. However, the GO coating layer


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could protected lipase from unfolding and denaturation through π-π stacking interaction with lipase. Moreover, GO edges possessing negatively charged carboxylate groups that can bind lipase via electrostatic interactions, the hydrophobic centers of GO nanosheets can also facilitate the opening of lipase lid and dispersion interaction-driven binding of enzymes. In particularly, lipase buried in GO sheets may cause mass transfer limitation that substrate may less accessible to active center leading lower activity. However, the using of GO coated lipase GO/PPL-IL-MCS may gain long-term stability and recovery. The good stability and recovery makes the industrial application of immobilized lipase much economic.

Figure 5. Effect of (a) pH and (b) temperature on free and immobilized lipase activity. The pH and temperature play important roles on the activity of lipase. Thus, the effect of pH was investigated in the range of 5.5 to 8.5 (Figure 5a) and the effect of temperature was studied in the range of 35 to 65 C (Figure 5b). As shown in Fig. 5a, the optimum pH for free PPL was 7.0 while the optimum pH for PPL-MCS and PPLIL-MCS were both 7.5, for GO/PPL-IL-MCS was 8.0, respectively. The shift of optimum pH for PPL-IL-MCS could be attributed to the introduction of ionic liquids which affected the surface charge of supports and immobilized lipase28. After coated with GO, the optimum pH for GO/PPL-IL-MCS shifted to 8.0 from 7.5, this indicated that GO coating make immobilized lipase more stable at higher pH. When the pH of reaction system was 8.5, the residual activity of GO/PPL-IL-MCS retained 88.3% which was higher than that of other three samples. Figure 5b showed that the optimum temperature for free PPL was 45 C and for the three kind of immobilized lipase both



were 50 C. The immobilized lipase exhibited good endurance to higher temperature. When temperature up to 65 C, the residual activity of PPL-MCS, PPL-IL-MCS and GO/PPL-IL-MCS was 66.9%, 72.6% and 88.5% respectively, while the free lipase retained only 45.7% of its initial activity. The improvement may attributed to that the introduction of ionic liquids improved the microenvironment of lipase and the protection of GO coating strengthened the conformational integrity of lipase. Stabilities of Immobilized Lipase

Figure 6. Stability performance of immobilized lipase. Thermal stability of immobilized enzymes plays an important role in their application. To study the thermal stability of immobilized lipase, the samples were incubated in in phosphate buffer at 50 C for different time as shown in Figure 6a. When incubation time increased, the activity of PPL-MCS, PPL-IL-MCS and GO/PPL-IL-MCS both decreased. The difference between the three samples was that the activity of PPL-MCS and PPL-IL-MCS decayed more rapidly than GO/PPL-IL-MCS with time increased. After incubated for 6 h, the residual activity for PPL-MCS, PPL-IL-MCS and GO/PPLIL-MCS was 40.6%, 62.7% and 73.4%, respectively. The introduction of IL can maintain the structural integrity and rigidity of enzyme which protected the lipase from unfolding at high temperature. Moreover, the GO coating shielded the enzyme from


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denaturation through π-π interaction which may anchor the lipase firmly. The results proved that the thermal stability of immobilized lipase was better after coated with GO nanosheets. Denaturants can cause enzyme unfolding, which significantly influence the industrial application of enzymes42. In order to investigate the stability of immobilized lipase in the presence of denaturants, immobilized lipase was incubated in urea solution with different concentration for 2 h. As shown in Figure 6b, with the increase of urea concentration, the activity of all samples decreased. When the urea concentration was 5M, PPL-MCS, PPL-IL-MCS and GO-PPL-IL-MCS preserved 56.5%, 72.8% and 90.6% of its initial activity, respectively. Urea can form H-bonds with lipase causing the conformation changes. When PPL immobilized on IL-MCS, the imidazole rings of IL and active groups of CS can form H-bonds with urea that protected the enzyme from unfolding to some extent. After coated with GO, the GO shell formed H-bonds with urea firstly that decreased the diffusion coefficient of urea and hindered their accessibility to the protein domains. The results confirmed that the GO shell can for further improve the tolerance of immobilized lipase to denaturants. Better reusability of immobilized enzymes can enhance their industrial application value. The major purpose of immobilization is to obtain ease in recovery and reusability of biocatalysts. Figure 6c showed the reusability of PPL-MCS, PPL-IL-MCS and GO/PPL-IL-MCS. After 10 cycles, the residual activity of GO-PPL-IL-MCS was 92.1% which was higher than that of PPL-MCS (72.6%) and PPL-IL-MCS (82.4%). This result indicated that the reusability of immobilized lipase was improved after coated with GO, this may due to the interaction of GO with lipase which may cause the open conformation of lipase. Furthermore, the GO coating can decrease the release of lipase through interaction with enzyme. The reusability of immobilized lipase in this work is also better than many reported samples29, 31. Figure 6d showed the storage stability of immobilized lipase for 35 days at 4 C. The activity of both immobilized lipase decreased with the storing time increasing. Obviously, the activity of GO/PPL-IL-MCS decreased more slowly than that of PPLMCS and PPL-IL-MCS. After 35 days, GO/PPL-IL-MCS preserved 87.5% of its initial



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activity, while PPL-MCS and PPL-IL-MCS retained 68.8% and 72.8% respectively. The results showed that GO coating can improve the storage stability of immobilized lipase. It can be attributed to that the interaction between GO and lipase reduced the conformational flexibility inhibiting the unfolding and denaturation of immobilized lipase. Kinetic Measurements Table 2. Kinetic parameters

apparent Km (mg/ml)

Vmax (U/mg)

PPL-MCS

17.1 ± 0.9

1.074 ± 0.06

PPL-IL-MCS

5.7 ± 0.5

3.27 ± 0.13

GO/PPL-IL-MCS

8.8 ± 0.7

2.93 ± 0.08

Samples

The kinetic parameters of immobilized lipase were investigated by measuring the activity of lipase with various substrate concentrations in PBS. The apparent Km and Vmax valuse were calculated from the Lineweaver-Burk plots and the results were shown in Table 2. The apparent Km of PPL-IL-MCS was 5.7 mg/ml lower than that of PPL-MCS (17.1 mg/ml). The decresing in apparent Km value indicated that PPL-ILMCS psrsented higher affinity between lipase and substrabe and had resonably high catalytic efficency43. The results could be owning to the introduction of IL causing a posivite conformation (lid opening ) of immobilized lipase leading to more accessible active sites. After coated with GO, the apparent Km of GO/PPL-IL-MCS increased to 8.8 mg/ml and Vmax decresed. This may ascribe to diffusion limitations and steric hindraces casusing by GO nanosheets, though hydrophobic centers of GO nanosheets can cause the opening of lipase lid through interface interaction. However, the introduction of GO coating enhanced the stability and recovery. CD Analysis Table 3. Percentages of secondary structure of enzymes Samples

α-Helix (%)

β-Sheet (%)

β-Turn (%)

Random coil (%)

PPL

29.8

26.5

18.7

26.4

PPL-MCS

26.7

28.6

18.2

26.5


Page 17 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


PPL-IL-MCS

18.4

35.3

16.6

29.7

GO/PPL-IL-MCS

17.1

40.4

17.3

25.2

Circular dichroism spectra was used to study the secondary structure of free and immobilized lipase as shown in Figure S5. In far-UV CD spectra (190-250 nm), it could identify the changes of α-helix, β-sheet, β-turn and random coil in enzyme44, 45. The secondary structure percentages of lipases were calculated from the CD spectra and summarized in table 3. All the immobilized lipase presented lower α-helix content but higher β-sheet compared to free lipase. A decrease in α-helix content promotes higher tendency for the open conformation of lipase which facilitate substrate access to active site. An increase in β-sheet content enhances the rigidity of lipase which leads to high thermal stability, denaturant tolerance. For PPL-IL-MCS, the introduction of IL enhanced the hydrophobicity of IL-MCS and the hydrophobic support binding with hydrophobic lid of lipase could disrupt lid’s helical structure leading to the open conformation. Furthermore, after coated with GO nanosheets, the GO/PPL-IL-MCS exhibited lowest α-helix content and highest β-sheet content and this may due to the GO coating can anchor the lipase throug π-π stacking interaction and interfacial interaction between hydrophobic center of GO sheets and helical lid of lipase. The results was in accordance with the activity assay and stability investigation. CONCLUSIONS In this work, ionic liquid modified magnetic chitosan nanoparticles were prepared and applied as supports for PPL immobilization. The functional group and alkyl chain length of ionic liquids had a significant effect on the enzymatic properties of immobilized PPL on thus prepared support. GO nanosheet was used to coat the immobilized PPL for anchoring the lipase structure and enhancing the lipase stabilities. This strategy improved the stabilities of immobilized lipase for further. The reason for the improvement of enzymatic properties was explained by secondary structure analysis. Moreover, the immobilized lipase can be easily recovered by using an external magnetic field with no significant loss of activity. Considering, these excellent



attributes, the lipase immobilization strategy in this work may be utilized as a versatile method for other enzymes immobilization. ASSOCIATED CONTENT Supporting Information Preparation of magnetic nanoparticles, optimization of immobilization conditions and experimental details for additional characterizations (XRD patterns, magnetization curves, water contact angle, CD spectra and EDS spectra). AUTHOR INFORMATION Corresponding Authors Tel. /Fax: +86-25-58139942. *E-mail: [email protected] (H. Huang) Tel. /Fax: +86-25-83172094 *E-mail: [email protected] (Y. Hu) Notes The authors declare no competing financial interest. ACKNOWLEDGEMENTS This work was financially supported by the National Natural Science Foundation of China (No. 21676143), the Jiangsu Synergetic Innovation Center for Advanced BioManufacture, Qing Lan Project of Jiangsu Province, the Self-Owned Research Project from Stake Key Laboratory of Materials-Oriented Chemical Engineering (Grant No. ZK201603), Yunnan Provincial Department of Education Science Research Fund Project (2015Y435, 2018JS449). REFERENCES 1.

Sheldon, R. A.; Woodley, J. M., Role of Biocatalysis in sustainable chemistry.

Chem. Rev. 2018, 118 (2), 801-838, DOI 10.1021/acs.chemrev.7b00203. 2.

Sheldon, R. A.; van Pelt, S., Enzyme immobilisation in biocatalysis: why, what and

how. Chem. Soc. Rev. 2013, 42 (15), 6223-6235, DOI 10.1039/C3CS60075K. 3.

Sheldon, R. A.; Pereira, P. C., Biocatalysis engineering: the big picture. Chem. Soc.

Rev. 2017, 46 (10), 2678-2691, DOI 10.1039/C6CS00854B.


Page 18 of 23

Page 19 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


4.

Lian, X.; Fang, Y.; Joseph, E.; Wang, Q.; Li, J.; Banerjee, S.; Lollar, C.; Wang, X.;

Zhou, H., Enzyme–MOF (metal–organic framework) composites. Chem. Soc. Rev. 2017, 46 (11), 3386-3401, DOI 10.1039/C7CS00058H. 5.

Magner, E., Immobilisation of enzymes on mesoporous silicate materials. Chem.

Soc. Rev. 2013, 42 (15), 6213-6222, DOI 10.1039/C2CS35450K. 6.

Li, D.; Zhang, W.; Yu, X.; Wang, Z.; Su, Z.; Wei, G., When biomolecules meet

graphene: from molecular level interactions to material design and applications. Nanoscale 2016, 8 (47), 19491-19509, DOI 10.1039/C6NR07249F. 7.

Cipolatti, E. P.; Valério, A.; Henriques, R. O.; Moritz, D. E.; Ninow, J. L.; Freire,

D. M. G.; Manoel, E. A.; Fernandez-Lafuente, R.; de Oliveira, D., Nanomaterials for biocatalyst immobilization – state of the art and future trends. RSC Advances 2016, 6 (106), 104675-104692, DOI 10.1039/C6RA22047A. 8.

Khoshnevisan, K.; Vakhshiteh, F.; Barkhi, M.; Baharifar, H.; Poor-Akbar, E.; Zari,

N.; Stamatis, H.; Bordbar, A.-K., Immobilization of cellulase enzyme onto magnetic nanoparticles: Applications and recent advances. Mol. Catal. 2017, 442, 66-73, DOI 10.1016/j.mcat.2017.09.006. 9.

Lawson, B. P.; Golikova, E.; Sulman, A. M.; Stein, B. D.; Morgan, D. G.; Lakina,

N. V.; Karpenkov, A. Y.; Sulman, E. M.; Matveeva, V. G.; Bronstein, L. M., Insights into Sustainable Glucose Oxidation Using Magnetically Recoverable Biocatalysts. ACS Sustain. Chem. Eng. 2018, 6 (8), 9845-9853, DOI 10.1021/acssuschemeng.8b01009. 10. Xie, W.; Huang, M., Immobilization of Candida rugosa lipase onto graphene oxide Fe3O4 nanocomposite: Characterization and application for biodiesel production. Energy Convers. Manage. 2018, 159, 42-53, DOI 10.1016/j.enconman.2018.01.021. 11. Hosseini, S. H.; Hosseini, S. A.; Zohreh, N.; Yaghoubi, M.; Pourjavadi, A., Covalent immobilization of cellulase using magnetic poly(ionic liquid) support: improvement of the enzyme activity and atability. J. Agric. Food. Chem. 2018, 66 (4), 789-798, DOI 10.1021/acs.jafc.7b03922. 12. Jesionowski, T.; Zdarta, J.; Krajewska, B., Enzyme immobilization by adsorption: a review. Adsorption 2014, 20 (5), 801-821, DOI 10.1007/s10450-014-9623-y. 13. Correro, M. R.; Moridi, N.; Schützinger, H.; Sykora, S.; Ammann, E. M.; Peters,



E. H.; Dudal, Y.; Corvini, P. F.-X.; Shahgaldian, P., Enzyme Shielding in an Enzymethin and Soft Organosilica Layer. Angew. Chem. Int. Ed. 2016, 55 (21), 6285-6289, DOI 10.1002/anie.201600590. 14. Jiang, Y.; Zhai, J.; Zhou, L.; He, Y.; Ma, L.; Gao, J., Enzyme@silica hybrid nanoflowers shielding in polydopamine layer for the improvement of enzyme stability. Biochem. Eng. J. 2018, 132, 196-205, DOI 10.1016/j.bej.2018.01.028. 15. Wang, C.; Luan, J.; Tadepalli, S.; Liu, K.-K.; Morrissey, J. J.; Kharasch, E. D.; Naik, R. R.; Singamaneni, S., Silk-Encapsulated Plasmonic Biochips with Enhanced Thermal Stability. ACS Appl. Mater. Inter. 2016, 8 (40), 26493-26500, DOI 10.1021/acsami.6b07362. 16. Chen, D.; Feng, H.; Li, J., Graphene Oxide: preparation, functionalization, and electrochemical applications. Chem. Rev. 2012, 112 (11), 6027-6053, DOI 10.1021/cr300115g. 17. Chen, X.; Wang, Y.; Zhang, Y.; Chen, Z.; Liu, Y.; Li, Z.; Li, J., Sensitive electrochemical aptamer biosensor for dynamic cell surface N-Glycan evaluation featuring multivalent recognition and signal amplification on a dendrimer–graphene electrode interface. Anal. Chem. 2014, 86 (9), 4278-4286, DOI 10.1021/ac404070m. 18. Zhang, J.; Zhang, F.; Yang, H.; Huang, X.; Liu, H.; Zhang, J.; Guo, S., Graphene Oxide as a Matrix for Enzyme Immobilization. Langmuir 2010, 26 (9), 6083-6085, DOI 10.1021/la904014z. 19. Zhang, Q.; Qiao, Y.; Hao, F.; Zhang, L.; Wu, S.; Li, Y.; Li, J.; Song, X.-M., Fabrication of a biocompatible and conductive platform based on a single-stranded DNA/Graphene nanocomposite for direct electrochemistry and electrocatalysis. Chem. –Eur. J. 2010, 16 (27), 8133-8139, DOI 10.1002/chem.201000684. 20. Mathesh, M.; Luan, B.; Akanbi, T. O.; Weber, J. K.; Liu, J.; Barrow, C. J.; Zhou, R.; Yang, W., Opening lids: modulation of lipase immobilization by graphene oxides. ACS Catal. 2016, 6 (7), 4760-4768, DOI 10.1021/acscatal.6b00942. 21. Itoh, T., Ionic liquids as tool to improve enzymatic organic synthesis. Chem. Rev. 2017, 117 (15), 10567-10607, DOI 10.1021/acs.chemrev.7b00158. 22. Liu, Y.; Shi, L.; Wang, M.; Li, Z.; Liu, H.; Li, J., A novel room temperature ionic


Page 20 of 23

Page 21 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


liquid sol–gel matrix for amperometric biosensor application. Green Chem. 2005, 7 (9), 655-658, DOI 10.1039/B504689K. 23. Suo, H.; Gao, Z.; Xu, L.; Xu, C.; Yu, D.; Xiang, X.; Huang, H.; Hu, Y., Synthesis of functional ionic liquid modified magnetic chitosan nanoparticles for porcine pancreatic lipase immobilization. Mater. Sci. Eng. C 2019, 96, 356-364, DOI 10.1016/j.msec.2018.11.041. 24. Itoh, T.; Nishimura, Y.; Ouchi, N.; Hayase, S., 1-Butyl-2,3-dimethylimidazolium tetrafluoroborate: the most desirable ionic liquid solvent for recycling use of enzyme in lipase-catalyzed transesterification using vinyl acetate as acyl donor. J. Mol. Catal. BEnzym. 2003, 26 (1), 41-45, DOI 10.1016/S1381-1177(03)00147-4. 25. Zhang, S.; Zhang, J.; Zhang, Y.; Deng, Y., Nanoconfined ionic liquids. Chem. Rev. 2017, 117 (10), 6755-6833, DOI 10.1021/acs.chemrev.6b00509. 26. Xie, W.; Zang, X., Lipase immobilized on ionic liquid-functionalized magnetic silica composites as a magnetic biocatalyst for production of trans-free plastic fats. Food Chem. 2018, 257, 15-22, DOI 10.1016/j.foodchem.2018.03.010. 27. Noori, A. R.; Hosseinkhani, S.; Ghiasi, P.; Akbari, J.; Heydari, A., Magnetic nanoparticles supported ionic liquids improve firefly luciferase properties. Appl. Biochem. Biotechnol. 2014, 172 (6), 3116-3127, DOI 10.1007/s12010-014-0730-8. 28. Suo, H.; Xu, L.; Xu, C.; Chen, H.; Yu, D.; Gao, Z.; Huang, H.; Hu, Y., Enhancement of catalytic performance of porcine pancreatic lipase immobilized on functional ionic liquid modified Fe3O4-Chitosan nanocomposites. Int. J. Biol. Macromol. 2018, 119, 624-632, DOI 10.1016/j.ijbiomac.2018.07.187. 29. Shao, Y.; Jing, T.; Tian, J.; Zheng, Y., Graphene oxide-based Fe3O4 nanoparticles as a novel scaffold for the immobilization of porcine pancreatic lipase. RSC Advances 2015, 5 (126), 103943-103955, DOI 10.1039/C5RA19276E. 30. Zhu, Y.; Ren, X.; Liu, Y.; Wei, Y.; Qing, L.; Liao, X., Covalent immobilization of porcine

pancreatic

lipase

on

carboxyl-activated

magnetic

nanoparticles:

Characterization and application for enzymatic inhibition assays. Mater. Sci. Eng. C 2014, 38, 278-285, DOI 10.1016/j.msec.2014.02.011. 31. Zou, B.; Song, C.; Xu, X.; Xia, J.; Huo, S.; Cui, F., Enhancing stabilities of lipase



Page 22 of 23

by enzyme aggregate coating immobilized onto ionic liquid modified mesoporous materials. Appl. Surf. Sci. 2014, 311, 62-67, DOI 10.1016/j.apsusc.2014.04.210. 32. Zang, L.; Qiu, J.; Wu, X.; Zhang, W.; Sakai, E.; Wei, Y., Preparation of Magnetic Chitosan Nanoparticles As Support for Cellulase Immobilization. Ind. Eng. Chem. Res. 2014, 53 (9), 3448-3454, DOI 10.1021/ie404072s. 33. Bradford, M. M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976, 72 (1), 248-254, DOI 10.1016/0003-2697(76)90527-3. 34. Li, L.; Wang, F.; Shao, Z.; Liu, J.; Zhang, Q.; Jiao, W., Chitosan and carboxymethyl cellulose-multilayered magnetic fluorescent systems for reversible protein

immobilization.

Carbohydr.

Polym.

2018,

201,

357-366,

DOI

10.1016/j.carbpol.2018.08.088. 35. Chen, H.; Cui, S.; Zhao, Y.; Zhang, C.; Zhang, S.; Peng, X., Grafting Chitosan with Polyethylenimine in an Ionic Liquid for Efficient Gene Delivery. Plos one 2015, 10 (4), e0121817, DOI 10.1371/journal.pone.0121817. 36. Wang, Z.; Zheng, L.; Li, C.; Zhang, D.; Xiao, Y.; Guan, G.; Zhu, W., A novel and simple procedure to synthesize chitosan-graft-polycaprolactone in an ionic liquid. Carbohydr. Polym. 2013, 94 (1), 505-510, DOI 10.1016/j.carbpol.2013.01.090. 37. Wan, X.; Xiang, X.; Tang, S.; Yu, D.; Huang, H.; Hu, Y., Immobilization of Candida antarctic lipase B on MWNTs modified by ionic liquids with different functional

groups.

Colloid.Surface.

B

2017,

160,

416-422,

DOI

10.1016/j.colsurfb.2017.09.037. 38. Wang, J.; Zhao, G.; Jing, L.; Peng, X.; Li, Y., Facile self-assembly of magnetite nanoparticles on three-dimensional graphene oxide–chitosan composite for lipase immobilization. Biochem. Eng. J. 2015, 98, 75-83, DOI 10.1016/j.bej.2014.11.013. 39. Patel, S. K. S.; Choi, S. H.; Kang, Y. C.; Lee, J.-K., Eco-Friendly Composite of Fe3O4-Reduced Graphene Oxide Particles for Efficient Enzyme Immobilization. ACS Appl.Mater. Inter. 2017, 9 (3), 2213-2222, DOI 10.1021/acsami.6b05165. 40. Rodrigues, R. C.; Ortiz, C.; Berenguer-Murcia, Á.; Torres, R.; Fernández-Lafuente, R., Modifying enzyme activity and selectivity by immobilization. Chem. Soc. Rev. 2013,


Page 23 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


42 (15), 6290-6307, DOI 10.1039/C2CS35231A. 41. Santos, J. C. S. d.; Barbosa, O.; Ortiz, C.; Berenguer-Murcia, A.; Rodrigues, R. C.; Fernandez-Lafuente, R., Importance of the Support Properties for Immobilization or Purification

of

Enzymes.

ChemCatChem

2015,

7

(16),

2413-2432,

DOI

10.1002/cctc.201500310. 42. Rabbani, G.; Ahmad, E.; Zaidi, N.; Fatima, S.; Khan, R. H., pH-Induced molten globule state of rhizopus niveus lipase is more resistant against thermal and chemical denaturation than its native state. Cell Biochem. Biophys. 2012, 62 (3), 487-499, DOI 10.1007/s12013-011-9335-9. 43. Royvaran, M.; Taheri-Kafrani, A.; Landarani Isfahani, A.; Mohammadi, S., Functionalized superparamagnetic graphene oxide nanosheet in enzyme engineering: A highly dispersive, stable and robust biocatalyst. Chem. Eng. J. 2016, 288, 414-422, DOI 10.1016/j.cej.2015.12.034. 44. Liu, T.; Liu, Y.; Wang, X.; Li, Q.; Wang, J.; Yan, Y., Improving catalytic performance of Burkholderia cepacia lipase immobilized on macroporous resin NKA. J. Mol. Catal. B-Enzym. 2011, 71 (1), 45-50, DOI 10.1016/j.molcatb.2011.03.007. 45. Jia, R.; Hu, Y.; Liu, L.; Jiang, L.; Zou, B.; Huang, H., Enhancing Catalytic Performance of Porcine Pancreatic Lipase by Covalent Modification Using Functional Ionic Liquids. ACS Catal. 2013, 3 (9), 1976-1983, DOI 10.1021/cs400404f. Table of Contents Graphic

The immobilized lipase exhibited excellent enzymatic propeties through a facile immobilzation method.


Graphene Oxide Nanosheets Shielding of Lipase Immobilized on

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