Covalent immobilization of lipase onto chitosan-mesoporous silica hybrid nanomaterials by carboxyl functionalized ionic liquids as the coupling agent

Accepted Manuscript Title: Covalent immobilization of lipase onto chitosan-mesoporous silica hybrid nanomaterials by carboxyl functionalized ionic liquids as the coupling agent Authors: Xinran Xiang, Hongbo Suo, Chao Xu, Yi Hu PII: DOI: Reference:

S0927-7765(18)30108-5 https://doi.org/10.1016/j.colsurfb.2018.02.033 COLSUB 9173

To appear in:

Colloids and Surfaces B: Biointerfaces

Received date: Revised date: Accepted date:

22-11-2017 18-1-2018 14-2-2018

Please cite this article as: Xinran Xiang, Hongbo Suo, Chao Xu, Yi Hu, Covalent immobilization of lipase onto chitosan-mesoporous silica hybrid nanomaterials by carboxyl functionalized ionic liquids as the coupling agent, Colloids and Surfaces B: Biointerfaces https://doi.org/10.1016/j.colsurfb.2018.02.033 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Covalent immobilization of lipase onto chitosan-mesoporous silica hybrid nanomaterials by carboxyl functionalized ionic liquids as the coupling agent

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State Key Laboratory of Material-Oriented Chemical Engineering, School of Pharmaceutical

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Xinran Xianga, Hongbo Suoa, Chao Xua, Yi Hua *

Sciences, Nanjing Tech University, Nanjing 210009, China

*Corresponding author at: Nanjing Tech University, No. 5 Xinmofan Road,

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E-mail address: [email protected] (Y. Hu).

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Nanjing 210009, China. Tel.: +86 25 83172094; fax: +86 25 83172094.

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Graphical abstract

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Highlights: 

Functional ionic liquid as a linker successfully prepared nano-composite carrier CTS-SBA-15

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Biocompatible, inorganic-organic nanocomposites and cross-linker used for immobilization Enzymatic properties of PPL immobilized on nanocomposites were improved

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

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obviously

Abstract Chitosan-mesoporous silica SBA-15 hybrid nanomaterials (CTS-SBA-15) were synthesized by means of carboxyl functionalized ionic liquids as the coupling

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agent. The as-prepared CTS-SBA-15 support was characterized by TEM, FTIR, TG and nitrogen adsorption-desorption techniques. Porcine pancreas lipase (PPL) was

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then bound to the hybrid nanomaterials by using the cross-linking reagent

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glutaraldehyde (GA). Further, the parameters like cross-linking concentration, time

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and ratio of supports to enzyme were optimized. The property of immobilized lipase were tested in detail by enzyme activity assays. The results indicated that the hybrid

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nanomaterials could form three-dimensional (3D) structure with homogeneous mesoporous structures and immobilized PPL revealed excellent enzymatic

Lipase, Enzyme immobilization, Nanomaterial, Covalent cross-linking,

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Keywords

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performance.

Chitosan, Ionic liquids

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1. Introduction

As highly effective, versatile and ecofriendly catalysts, enzyme is widely

recognized as potentially the first choice in industrial-scale applications. With the widespread use of lipase in the industry, people put a higher demand for the performance of lipase, such as high catalytic activity, stability and reusability, but free lipase is difficult to fully meet these requirements, it is necessary to immobilize the 2

lipase to provide inexpensive, well-behaved lipase preparations for industrial applications. Porcine pancreas lipase (PPL) is one of the most widely used lipases and is cheaper compared with other commercial microbial and animal lipases [1]. It can not only catalyze the hydrolysis of oil, but also catalyze ester synthesis reaction, transesterification, acid hydrolysis reaction, etc [2, 3]. Nevertheless, the industrial applicability of PPL in the native form is always obstructed due to low operational

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stability, difficulty in recovery and reusability [4].

Enzyme immobilization on solid supports is the most important route that can

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overcome these drawbacks and improve the properties of the biocatalyst. Therefore, enzyme immobilization is undertaken either for the purpose of basic research or for use in technical processes of industrial manufacture. At present, the research on

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enzyme immobilization technology is mainly divided into supports and methods of

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immobilization. A variety of synthetic inorganic or organic materials can be used as

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supports, and enzyme can be immobilized by physical adsorption, cross-linking, metal chelation or covalent binding on carrier surface [5-7]. Since the actual use of each

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enzyme must be carefully selected and dependent on the selected enzyme, the type of

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support and method immobilization are of fundamental importance for the successful application of the biocatalyst. Glutaraldehyde is the preferred cross-linker due to its inexpensive, easily available, easy to manipulate and has the ability to make covalent

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bonds with most enzyme [8].

In recent years, nanostructured composites combined the advantages of inorganic

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materials and natural polymer materials have become one of the most efficient supports for enzyme immobilization and gained more and more attention because of their unique properties [9, 10]. Mesoporous materials such as SBA-15 have the

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advantage of being enzyme-immobilized carriers : large specific surface area and pore volume, uniform distribution of nano-sized pores, rich hydroxyl groups on the surface and inertness. Hence, the assembly of biological macromolecules in mesoporous materials has become the international hot spots of research on mesoporous materials. Chitosan from naturally occurring sources is a biocompatible natural hydrophilic cationic polysaccharide (pKa 6.3-7.0) that have unique structures, multidimensional 3

properties, highly sophisticated functionality and a wide range of applications in biomedical and other industrial areas [11, 12]. In particular, chitosan molecules contain free amino groups, through the cross-linking agents (such as glutaraldehyde) are prone to indirect covalent association with enzyme to make them firmly fixed to chitosan [13]. As designable green solvents, ionic liquids (ILs) have been widely studied in

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organic synthesis, electrochemistry, materials science, separation engineering and

other fields due to their low volatility, chemical stability [14]. Ionic liquids has

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showed great potential in the field of enzyme catalysis. Many kinds of enzyme in ionic liquids as a reaction medium or additive showed good activity, stability and selectivity [15, 16].

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In our previous study, modified nanocarriers (SBA-15 or carbon nanotubes) by

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functionalized ionic liquids were used to immobilize lipases to enhance their

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enzymatic properties [17, 18]. And glutaraldehyde was used as a crosslinking agent to further enhance the enzyme loading and immobilized enzyme stability [17].

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In this work, the nanostructured biocomposites were synthesized through the

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assembly of natural macromolecule material CTS onto nanosized mesoporous siliceous SBA-15 via functionalized ionic liquids as the novel coupling agent to not only protect the active site of imobilized enzyme but also improve the affinity with

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the substrate. And the use of glutaraldehyde as a cross-linking agent to further enhance the stability of immobilized enzyme and reusability. The different parameters

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like cross-linker concentration and cross-linking time were optimized. 2. Experimental section

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2.2 Carboxyl-Modification of inorganic mesoporous material SBA-15. SBA-15 was modified by minor modification of previous works [19, 20]. The

detailed process was described as follows: 2 g SBA-15 was dispersed in 100 mL toluene solution at room temperature, then 10 mmol CPTMO was added dropwise and reacted at 95 °C for 8 h under nitrogen atmosphere. The obtained solid was isolated by filtration and repeatedly washed with anhydrous ethanol and ether. Intermediate product and 10 mmol imidazole were mixed with 100 mL chloroform at 70 °C for 24 4

h, then filtered, washed with ether and Soxhlet-extracted with dichloromethane for 24 h. Alkylation reaction was done with product and chloroacetic acid for 12 h, then filtered , washed with ether and Soxhlet-extracted with dichloromethane for 24 h. The resultant solid and 2.5 mmol NaBF4 were suspended in 100 mL of acetone to complete ionic exchange for 72 h under atmosphere with constant stirring. The product was carboxyl-functionalized ionic liquids modified SBA-15 named

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COOH-SBA-15, which was washed with ether three times, Soxhlet-extracted with

2.3 Synthesis of CTS-SBA-15 hybrid nanomaterials.

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dichloromethane for 24 h and then subjected to application in the next step.

Conjugation of COOH-SBA-15 with CTS was accomplished via a covalent mechanism. The CTS-SBA-15 nanocomposite was prepared by the following method:

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2 g COOH-SBA-15 in phosphate buffer solution was sonicated for 2 h to obtain a

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homogeneous dispersion. Next, a solution of 0.05 M EDC and 0.05 M NHS were

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added to COOH-SBA-15 dispersion with continuous stirring for 2 h to activate the COOH groups of SBA-15. 3% CTS solution was prepared by dissolving 0.6 g CTS

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into aqueous solution of 20 mL acetic acid under ultrasonic stirring for 2 h at room

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temperature. Then the CTS solution was added dropwise to the activated COOH-SBA-15 solution within 30 min, and the reaction was performed at room temperature under sonication for 6 h. After additional reaction for 8 h, the mixture

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was centrifuged and washed with 0.1 M acetic acid solution and de-ionized water. The obtained product was denoted as CTS-SBA-15.

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2.4 Characterization

FT-IR spectra was obtained in transmission mode on a 170-SX Fourier-transform

infrared spectrophotometer in the range of 500-4000 cm-1 using the KBr pellet

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technique. The microstructures of the nanotubes were observed using a transmission electron

microscope

(TEM,

JEOL

JEM-2010,

200

KV).

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adsorption-desorption isotherms were measured on a Micromeritics ASAP 2020M surface area and porosity analyzer at 77 K. Pore size distribution was calculated by the Barrett-Joyner-Halenda (BJH) method. The Brauner-Emmet-Teller (BET) surface area and pore-size distribution of the SBA-15 and CTS-SBA-15 was calculated using 5

experimental points at a relative pressure of P/P0 =0.05-0.20. The total pore volume was calculated by the N2 amount adsorbed at the highest P/P0(P/P0 ≈0.99). 2.5 Immobilization of PPL and determination of enzyme loading In this study, PPL was immobilized on different supports by the strategy of covalent bonding. First, SBA-15 and amine-functionalized CTS-SBA-15 support were activated by GA activation procedure. GA activation procedure is as follows: an

solution

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appropriate amount of CTS-SBA-15 was respectively dispersed in 0.5-4.5% GA were introduced, then allowed to incubated for 3-24 h for shaking at 150

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rpm at room temperature. SBA-15 has been stepped above at 70℃ in pH 8.0 buffer solution. The activated support was taken out by centrifugal separation, washed several times with deionized water to remove excess GA. The obtained product was

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respectively denoted as GA-SBA-15 and GA-CTS-SBA-15.

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0.5 g supports (GA-SBA-15 or GA-CTS-SBA-15) were dispersed in 25mL pH 7.0

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phosphate buffer solution by sonication for 30 min. After being separated equably, the supports were placed into 4% (m/v) PPL solution, and then the immobilization

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process was performed at 35 ℃ on a shaking-table with rotational speed of 150 rpm

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for 2 h. Later, the immobilized PPL was recovered by centrifugal separation, and thoroughly rinsed three times with phosphate buffer solution to remove unbound PPL. The washed solution was collected to assay the amount of residual enzyme. The

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resulting immobilized PPL was dried in a freeze-dryer and stored at 4 ℃ prior to use. The immobilized PPL by GA-SBA-15 and GA-CTS-SBA-15 were remarked as

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PPL-SBA-15 and PPL-CTS-SBA-15 respectively. The enzyme loading is defined as the enzyme amount difference between the total

enzyme used and the residual enzyme present in the supernatant after immobilization.

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The enzyme loading was determined using the following formula: (Ci − Cf )V W where protein bound is the amount of bound enzyme onto CTS-SBA-15 hybrid protein bound(mg⁄g) =

nanomaterials (mg·g-1), Ci and Cf are the concentrations of the enzyme protein initial and final in the reaction medium (mg·mL-1), V is the volume of the reaction medium 6

(mL), and W is the weight of the carriers (g). All data used in this formula are the average of triplicate experiments. 2.6 Enzyme activity of PPL measurement The enzyme activity of the immobilized PPL can be determined by titrating acetic acid which is a by-product in the hydrolysis of triacetin. The immobilized PPL (0.1 g) was added to 25 mL of 4% (w/v) homogenized triacetin. The reaction mixture was

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incubated at 35 ℃ and pH 7.0 phosphate buffer on a reciprocal shaker at 110 rpm for

10 min. The mixture solution was then titrated with 0.05 mol/L NaOH solution to

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maintain pH 7.0. The volume of NaOH consumed was measured. One lipase unit (1 U) was defined as the amount of PPL that can consume 1 μmol triacetin per minute under assay conditions. A blank was determined first andanalys is followed. Enzyme

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activity was calculated from the quantity of NaOH added per unit time and expressed

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as unit/mg of solid matter for composites and unit/mg of the native enzyme. All

were calculated.

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2.7 Stability of the immobilized PPL

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measurement experiments were carried out at least three times and average values

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Stability as the most important performance index of immobilized enzyme in practical application included thermal stability, storage stability and reusability. Three kinds of performance was investigated through the determination of the residual

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activity towards the the triacetin hydrolysis reaction. In order to determine the storage stability behavior of immobilized enzyme, the

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immobilized PPL was stored at 4 ℃ in refrigerator for a period of one month. The activity of immobilized enzyme was checked as above procedure (35 ℃, pH 7.0) in six repeated cycles in every 5 days interval. The relative activity was defined as the

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ratio of the residual activity to the initial activity. The thermal stability of the immobilized enzyme was investigated by the

preincubation of free enzyme and immobilized enzyme at 60 ℃ and pH 7.0 for the same time interval, then the residual enzyme activity was measured. The reusability of the immobilized enzyme was investigated through the determination of the residual activity in the same conditions. In the standard 7

conditions, the mixture that was composed of immobilized enzyme and glyceryl triacetate emulsion was reacted at 35 °C and pH 7.0. After 10 min of reaction, the spent immobilized enzyme was recovered by centrifugal separation, washed three times with phosphate buffer of pH 7.0, then finally freeze-dried. The recovered immobilized enzyme was employed in the next group of triacetin hydrolysis reaction under the same reaction conditions.

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2.8 Kinetic parameters

Kinetics parameters (Km and Vmax) of free and immobilized PPL were determined

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using different triacetin concentrations in the range of 0.01–0.1 mg·mL-1 in phosphate buffer at 35 ℃. Km and Vmax values of free and immobilized PPL were calculated from by ploting the initial reaction rate to the various of triacetin emulsification

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solution according to the linearized form of Michaelis-Menten kinetic equation by the

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pH-stat.

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2.9 Secondary structure analysis of free and immobilized PPL The changes in secondary structure of free and immobilized PPL were examined by

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using circular dichroism (CD). The PPL incubated at 35 °C and 60 °C for 30 min. The

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CD spectra of free PPL and immobilized PPL were noted by using CD from 180 to 300 nm with sample dispersed in the phosphate buffer. Using the corresponding supports as the blank, the samples were quantitatively tested with a circular

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dichromatograph (JASCO- J810). The optical range of the quartz sample pool is 1cm; the range of scanning measurement wavelength is 180-300nm and the scanning speed

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is 50nm·min-1. Each sample was scanned 10 times and then averaged[21].

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Fig. 1: Schematic illustration of the synthesis process used to produce hybrid nanomaterials

3. Results and discussion

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3.1 Characterization of immobilized supports

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CTS-SBA-15 and changes in the structure of mesopores.

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Fig. 1 showed the synthesis strategy of SBA-CIL-CS and changes in pore structure

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of mesoporous materials. The morphologies of SBA-15 were characterized by TEM. The image of TEM shows that the HNTs have clearly two-dimensional six-party

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longitudinal structure and long-range mesopore channel structure (Fig. 2a). The SBA-15 are open at the tow ends. And no particles or other nanostructures were found

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in the TEM observation, indicating that the product consists of pure nanomaterials and the surfaces of nanomaterials are smooth and the edges are clear.

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From the COOH-SBA-15 (Fig. 2b), it could be clearly observed that a few transparent shadows appear in the orderly long-range structure of SBA-15 in contrast to pure SBA-15. The main structure of SBA-15 was still very clear. It was clearly

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indicated that the modification of carboxyl-functionalized ionic liquids onto the surface of SBA-15 did not destroy the long-range mesopore channels and Si-O-Si network of the support materials.

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Fig. 2: TEM a for SBA-15, COOH-SBA-15 and CTS-SBA-15.

The TEM of the dry composites (CTS-SBA-15) are shown in Fig. 2c. The hybrid

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nanomaterials formed three-dimensional (3D) nanocomposites with hierarchically porous structure. Compared with the natural SBA-15, the surface of CTS-SBA-15

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becomes rougher and less distinguished, namely thicker. The results of TEM images

confirmed that the as-synthesized products were successfully prepared. The modification with CTS created surface roughness which could in turn enhance the

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adhesion and create new surface area for enzyme.

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The FT-IR spectra in the 4000-450 cm-1 wavenumber range of SBA-15 (a),

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COOH-SBA-15 (b), CTS-SBA-15 (c) have been included in Fig. 3a. The infrared spectrum of SBA-15 showed characteristic peaks including the Si-O-Si antisymmetric

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stretching vibration (1095 cm−1), the symmetric stretch of Si-O (798, 955cm-1) and asymmetric stretch of O-H (3450 cm−1). As shown in Figure 3b, the peaks at 2950

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cm-1 and 1470 cm-1 were the C-H vibration of the aliphatic compound and the stretching vibration of imidazole rings, respectively. The absorption peaks at 1665

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cm-1 and 1570 cm-1 were respectively the stretching vibration of C=N and C=C groups of imidazole ring. For COOH-SBA-15 samples, the absorption peak of C=O in

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-COOH was around 1720 cm -1. These existing absorption peaks indicated that the carboxyl-functionalized ionic liquids has been successfully grafted onto the surface of SBA-15. Compared to the unmodified sample, CTS-SBA-15 samples exhibited some

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new peaks at 1442cm-1 (δC- H, amide I) and 1622cm-1 (amide II) [22]. Accordingly, the spectrum of CTS-SBA-15 samples contains both the characteristic peaks of raw HNTs and that of CTS, which indicated HNT was successfully combined with CTS [23]. FT-IR characterization demonstrated that SBA-15 and CTS were covalently combined by functional ionic liquids. The TG curves of SBA-15, COOH-SBA-15, and CTS-SBA-15were shown in Fig 10

3b. The original SBA-15 was stable below 800 ℃ and a slight weight loss (5.12%) of SBA-15 was due to the dehydration and/or dehydroxylation reactions on its surface [24]. However, ionic liquids and chitosan modified SBA-15 have showed significant weight loss in each region. The weight loss of the first region (350 ℃) might be the decomposition of the imidazolium salts grafted on the surface of SBA-15. The significant weight loss in the second region (680 ℃ ) might be due to the

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decomposition of chitosan itself, indicating that chitosan has been modified to the surface of SBA-15 by ionic liquids as the coupling agent.

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Fig 3(c, d) showed nitrogen adsorption-desorption isotherms and the corresponding pore size distributions of SBA-15 and CTS-SBA-15 samples, respectively. As shown in Fig 3(c, d), all isotherms were of typical type IV according to the International

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Union of Pure and Applied Chemistry (IUPAC) classification [25], and had H1

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hysteresis loops with the sharp adsorption and desorption branches in the P/P0 range

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of 0.6–1.0. The isotherms accounted for the relatively ordered mesoporous structure and the results also indicated that all samples had narrow pore size distributions [26].

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The Barrett-Joyner-Halenda (BJH) pore volume and the Brunauer-Emmett-Teller (BET) surface area of SBA-15 were were calculated by N2 adsorption-desorption

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isotherms. The SBA-15 sample exhibited a high BET surface area of 757.8 m2/g, pore volume of 0.94 cm3/g, and mean pore size of 6.6 nm. As for CTS-SBA-15, the

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measured surface area and pore volume were estimated to be 49 m2/g and 0.11 cm3/g, respectively. The results confirmed that ionic liquid and chitosan grafted on the

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surface of mesoporous material SBA-15 made the pore volume, diameter and BET

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surface area decrease, but it still retained its mesoporous structure.

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Fig. 3: (a) FT-IR spectrum and (b) TG curves of SBA-15, COOH-SBA-15, CTS-SBA-15;

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nitrogen adsorption desorption isotherms of (c) SBA-15 and (d) CTS-SBA-15.

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3.2 Immobilization of PPL on supports by organic cross-linker The porcine pancreatic lipase was covalently immobilized onto SBA-15 and

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CTS-SBA-15 by using GA as organic cross-linker. The reactive aldehyde groups of GA can cross-link amino groups on the surface of CTS-SBA-15 composites in terms

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of lysine groups of lipase and chitosan to form Schiff’s base which prevents leaching of enzyme during reaction [27]. Cross-linking parameters (cross-linker concentration

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and cross-linking time) were the key steps in immobilization which have direct influence on enzyme loading, activity and operational stability. The extent of cross-linking between enzyme and CTS-SBA-15 is needed to be optimized to get

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maximum activity recovery. The effect of cross-linker concentration on the activity recovery of PPL in immobilized form was studied by varying concentration in the range of 0.5-4.5% (v/v) (Fig 4a). The activity recovery of PPL decreased with increase in cross-linker concentration. The retained activity of immobilized PPL on CTS-SBA-15 at different GA concentrations was higher than that of SBA-15, and the trend of decrease was more gradual. At high concentration of cross-linker (1.5–4.5%), 12

less activity recovery was observed due to cross-linking of enzyme leading to the change for secondary structure of PPL. Further increase in the concentration of cross-linker resulted into higher rigidification of enzyme, The phenomenon resulted in the further destruction of the conformation of enzyme and the more loss of enzyme activity. The maximum activity recovery was found at 1.0% glutaraldehyde for SBA-15 and 1.5-2.0% for CTS-SBA-15.

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Cross-linking time is another important factor in immobilization of enzyme [28]. As Fig. 4 (b) shown, it can be seen that the maximum activity recovery in

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immobilized PPL on SBA-15 was found at 2 h of cross-linking time and that of CTS-SBA-15 was 3h. Further increase in cross-linking time, decreased the activity recovery of PPL gradually. This might be due to excessive cross-linking, which is

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speculated to cause the chemical modification of enzyme.

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To maximize the amount of PPL for immobilization, The ratio of supports and

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enzyme should be optimized. This was done by varying the ratio of supports to PPL (1:1–5:1) (Fig 4c). At lower ratio (1:1–3:1), the activity recovery of PPL was relative

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low .The reasons maybe that the amount of supports including SBA-15 and

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CTS-SBA-15 was not sufficient to load all number of PPL or that the immobilized PPL on the surface of supports is too dense and causes the substrate to interact with the active site of PPL well. As the ratio increased, the activity recovery also increased

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significantly up to up to 95% till the ratio of 4:1. With further increase in the supports to enzyme ratio, the activity recovery remained constant, and it might be due to the

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optimum loading of enzyme on supports [29]

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Fig. 4: Effect of cross-linker concentration (a), cross-linking time (b) and ratio of supports to enzyme (w/w) (c) on activity recovery of PPL. *

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The 100% activity recovery corresponds to PPL (340 U/g). The measurements were

performed in triplicate and the error bar represents the percentage error.

3.3 Catalytic Property of Immobilized PPL Table 1 showed the enzyme loading and activity of PPL immobilized on SBA-15 and CTS-SBA-15 samples. By adjusting the concentration of enzyme solution from 5 to 35 mg/ml (Fig 6a), the maximum of enzyme loading of the two immobilized 14

carriers SBA-15 and CTS-SBA-15were obtained. As shown in Table 1, the enzyme loading of CTS-SBA-15 were obviously higher than SBA-15. That CS uniformly distributed on the surface of SBA-15 took full advantage of the excellent properties of CS as a natural macromolecular material and solved the problem of low efficiency and biocompatibility for immobilized enzyme on SBA-15, and the covalent attachment amount of PPL molecules increased from 165.8 to 258.7 mg/g. Compared

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to pure SBA-15, inorganic and organic nanomaterials CTS-SBA-15 not only showed

a significantly higher immobilization efficiency, but also maintained the relatively

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high activity. Compared to the free enzyme, the relative activity of PPL-CTS-SBA-15 increased to 3.3 times, and 2.1 times than that of SBA-PPL. This phenomenon could be attributed that these inorganic immobilization carriers equipped natural ingredients

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via functional ionic liquids can protect the secondary structure at active site of PPL

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and decrease conformation change of enzyme during the immobilization process to

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ensure the stability of 3D structure of PPL [30]. It’s worth to note that PPL-CTS-SBA-15 showed higher activity and immobilization efficiency than those

immobilization [33, 34].

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reported in our previous work [17, 19, 31, 32] and some other new literatures for PPL

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Table 1. Activity and Kinetic Parameters of immobilized PPL*

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performance of Immobilized enzyme

biocatalysts

Protein loading

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(mg·g-1)

kinetic parameter

relative Vmax

Expressed

Km activity

(mg/min·g

Vmax/Km (mg/mL)

activity (U.g-1)

PPL) (%)

PPL-SBA-15

165.8

90.3

544.6

187.4

33.2

5.6

PPL-CTS-SBA-15

258.7

289.2

1117.9

576

20.4

28.2

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*The protein content of lipase is 17%, and the activity of free PPL is 340 U/g PPL (using triacetin at pH 7.0 and 35°C)

3.4 Kinetic parameters The activities of the immobilized PPL were plotted in the form of [S]-V plots, as shown in Fig 5, and Kinetic parameters (Vmax and Km) values were calculated from the intercepts on x- and y-axes, respectively. The kinetic behaviors of immobilized PPL on SBA-15 and CTS-SBA-15 were compared (Fig. 5), and the values of Vmax and 15

Km are summarized in Table 1. The apparent kinetic parameter Km for PPL-CTS-SBA-15 was lower than that for PPL-SBA-15, demonstrating the increase in

enzyme-substrate

affinity

of

PPL-CTS-SBA-15.

The

increase

of

the

enzyme-substrate affinity of CTS-SBA-15 indicated by the decrease of the Km value and increase of the Vmax values was possibly be attributable to one or more of the following reasons: (1) This could be probably that biomacromolecule CTS protected

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conformational flexibility even after cross-linking which maintained accessibility for

macromolecular substrate and helped the enzyme to orient suitably its active site

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toward the substrate, which is in accordance with a previous report; (2) Organic cross-linker covalently binds lipase along its linear chain. This prevents lump formation of enzyme molecules, resulting in unchanged mass transfer of substrate and

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product, which can be supported by similar studies [35].

Fig. 5: Scatchard plot of kinetic parameters of PPL-SBA-15 and PPL-CTS-SBA-15

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3.5 Stability study of free and immobilized PPL For enzyme immobilization, critical factors needed to be taken into account is the

operational stability of immobilized enzyme including thermal, storage stability and reusability. Thermal stability is a fundamental property of biocatalysts especially for their application in industry. As shown in Fig. 6b, it suggested that the thermal stabilities of the PPL immobilized on hybrid-nanomaterials CTS-SBA-15 were 16

significantly increased. Incubating at 70 ℃ for 7 hours, the residual activity of the immobilized lipase of CTS-SBA-15 still retained more than 60%, while that of SBA 15 lost almost activity. This phenomenon could be attributed to the reason that the introduction of chitosan improved the microenvironment of immobilized enzyme, so that it could protect enzyme conformations from destruction by high temperature. It seems that covalent cross-linking between amino residues present on the surface of

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lipase and CTS-SBA-15 via glutaraldehyde maintained the active tertiary structure of enzyme in immobilized form against native forms [36].

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As shown in Fig. 6c, under the same storage condition, the activity of PPL-SBA-15 dropped much faster than that of PPL-CTS-SBA-15. The immobilized PPL on CTS-SBA-15 retained about 70% of its original activity in the buffer solution after 30

U

day. In contrast, the PPL-SBA-15 only maintained about 36% of its original activity

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over the same period of time.

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Unlike free enzyme, immobilized enzyme could be easily separated from reaction system and reused, therefore reusability of immobilized enzyme is a key factor to

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make process economically feasible under practical application. The residual activity

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of immobilized PPL on CTS-SBA-15 was found to be 85% even after ten consecutive cycles of reuse and that of SBA-15 not enough 40% was maintained (Fig. 6d). The immobilized lipase was not leaching in the reaction mixture after multiple cycles of

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use which indicates significant reduction. The reason of reduction in residual activity might be due mechanical damage, deactivation of lipase and substrate inhibition due

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to the accumulation of the reaction products covered the surface of enzyme during

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recycling.

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Fig. 6: Properties of immobilized PPL: (a) Effect of the concentration of PPL on the enzyme loadings of SBA-15 and CTS-SBA-15. (b) Thermal stability of immobilized PPL at 70 ℃. (c) Storage stability

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of immobilized PPL and (d) Reuse capacity of immobilized PPL.

3.6 Secondary structure analysis of free and immobilized PPL

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Circular dichroism spectra is one of the most widely used techniques for determining the structure of proteins, making it possible to quantify conformational

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modifications in the 3D structure from changes. The secondary structures from the CD spectra were computed using the Jwsse32 software following the method of CD-Yang.Jwr (Table 2). The secondary structure distribution of native enzyme was

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determined from an aqueous solution at 35 °C, which indicates that the α-helix content agrees with the reported data [37]. According to the CD data analysis (Table 2A),all the immobilized PPL lower α-helix content but higher β-sheet content in comparison with free PPL. However, it was found that the PPL-CTS-SBA-15 had a relatively higher ratio of α-helix compared with PPL-SBA-15. The higher ratio of α-helix demonstrated better retention of 3D structure of lipase [17]. The analysis of 18

the CD spectrum of thesamples incubated at 60 °C for 30 min (Table 2B) showed a decline in both α-helix and β-sheet secondary structures, which agree with an unfolded protein state. This loss of native enzyme conformation is clearly correlated with poor thermostability, as shown in Table 2. Enzyme immobilization preserves catalytic activity, which is reflected by the maintenance of the secondary structure of PPL. Compared with free PPL and PPL-SBA-15, PPL-CTS-SBA-15 showed a

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significant increase in the ratio of β-sheet, indicating that the rigidity of PPL got a

certain degree of enhancement and its activity and stability was improved. These

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results was consistent with the experimental data of enzymatic performance test

showed that how the active enzyme conformation was protected by chitosan grafting against deactivation, displaying high stability .

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Table 2. Secondary Structure Percentages of Enzyme from CD Spectra: 35 °C without Incubation (A)

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and incubation at 60 °C for 30 min (B) Total beta-sheet(%)

Beta-turn (%)

Random coil (%)

PPL

27.3±0.3

28.2±0.7

17.2±0.4

27.4±0.6

PPL-SBA-15

15.1±0.5

32.3±0.2

16.6±0.3

32.7±0.5

PPL-CTS-SBA-15

22.2±0.6

40.3±0.4

16.0±0.3

22.4±0.7

20.4±0.7

24.6±0.2

40.3±0.4

11.4±0.2

30.8±0.1

11.5±0.4

43.7±0.5

20.2±0.3

37.3±0.6

13.6±0.3

29.4±0.8

PPL-SBA-15

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PPL-CTS-SBA-15

14.7±0.5

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PPL

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(B)

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(A)

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Alpha helix(%)

sample

4. Conclusions

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A new-style hybrid materials CTS-SBA-15 were prepared by chemical method via

ionic liquids as coupling agent and were used to immobilize PPL by organic cross-linker. The PPL immobilized on CTS-SBA-15 could exhibit higher immobilization yield and excellent properties of enzymology. The Vmax and Km values of immobilized PPL on CTS-SBA-15 were found to be improved obviously due to introduce chitosan to reserve conformational flexibility of enzyme even after 19

immobilization. Moreover, PPL-CTS-SBA-15 showed 85% residual activity even after 10 cycles and better thermal stability and storage stability which emphasizes its high stability and durability for industrial applicability. The immobilized PPL on CTS-SBA-15 exhibited overall high immobilization efficiency and excellent stability, which illustrates that hybrid materials CTS-SBA-15 should be promising in the field of immobilized enzyme.

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Acknowledgments

This work was financially supported by the National Natural Science Foundation of

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China (21676143) , Qing Lan Project of Jiangsu Province , the Natural Science

Foundation of Jiangsu Higher Education Institutions of China (14KJB530002), Program for Innovative Research Team in University of Jiangsu Province and

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Self-Owned Research Project from Key Laboratory of Material-Oriented Chemical

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Engineering (Grant No. ZK201603).

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References [1] A.A. Mendes, P.C. Oliveira, H.F. de Castro, Properties and biotechnological applications of porcine pancreatic lipase, J. Mol. Catal. B-Enzym. 78 (2012) 119-134. [2] C.H. Ali, A.S. Qureshi, S.M. Mbadinga, J. Liu, S. Yang, B. Mu, Biodiesel production from waste cooking oil using onsite produced purified lipase from Pseudomonas aeruginosa FW_SH-1: Central composite design approach, Renew.

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Energ. 109 (2017) 93-100.

[3] E. Doustkhah, S. Rostamnia, B. Gholipour, B. Zeynizadeh, A. Baghban, R.

SC R

Luque, Design of chitosan-dithiocarbamate magnetically separable catalytic

nanocomposites for greener aqueous oxidations at room temperature, Molecular Catalysis 434 (2017) 7-15.

U

[4] A. Madhavan, R. Sindhu, P. Binod, R.K. Sukumaran, A. Pandey, Strategies for

N

design of improved biocatalysts for industrial applications, Bioresource Technol. 245

A

(2017) 1304-1313.

[5] T. Jesionowski, J. Zdarta, B. Krajewska, Enzyme immobilization by adsorption: a

M

review, Adsorption 20 (2014) 801-821.

ED

[6] P. Lin, D. Weinrich, H. Waldmann, Protein biochips: oiented surface immobilization of proteins, Macromol. Chem. Phys. 211 (2010) 136-144. [7] Y. Liu, J. Yu, Oriented immobilization of proteins on solid supports for use in

PT

biosensors and biochips: a review, Microchim. Acta 183 (2016) 1-19. [8] F.B.H. Rehm, S. Chen, B.H.A. Rehm, Enzyme engineering for in situ

CC E

immobilization., Molecules 21.10(2016) 1370. [9] J.P. de Mesquita, C.L. Donnici, F.V. Pereira, Biobased nanocomposites from layer-by-layer assembly of cellulose nanowhiskers with chitosan, Biomacromolecules

A

11 (2010) 473-480. [10] M. Chen, G. Zeng, P. Xu, C. Lai, L. Tang, How do enzymes 'meet' nanoparticles and nanomaterials? Trends Biochem. Sci. 42 (2017) 914-930. [11] U. Klinkesorn, D.J. McClements, Influence of chitosan on stability and lipase digestibility of lecithin-stabilized tuna oil-in-water emulsions, Food Chem. 114 (2009) 1308-1315. 21

[12] I. Younes, M. Rinaudo, Chitin and chitosan preparation from marine sources. structure, properties and applications, Mar. Drugs 13 (2015) 1133-1174. [13] J.A. Camarero, Recent developments in the site-specific immobilization of proteins onto solid supports, Biopolymers 90 (2008) 450-458. [14] W. Gao, F. Zhang, G. Zhang, C. Zhou, Key factors affecting the activity and stability of enzymes in ionic liquids and novel applications in biocatalysis, Biochem.

IP T

Eng. J. 99 (2015) 67-84.

[15] H. Zhao, Protein stabilization and enzyme activation in ionic liquids: specific ion

SC R

effects, J. Chem. Technol. Biot. 91 (2016) 25-50.

[16] M. Bekhouche, L.J. Blum, B. Doumeche, Ionic liquid-inspired cations covalently bound to formate dehydrogenase improve its stability and activity in ionic liquids,

U

ChemCatChem 3 (2011) 875-882.

N

[17] B. Zou, Y. Hu, L. Jiang, R. Jia, H. Huang, Mesoporous material SBA-15

A

modified by amino Acid ionic liquid to immobilize lipase via ionic bonding and cross-Linking method, Ind. Eng. Chem. Res. 52 (2013) 2844-2851.

M

[18] Y. Hu, S. Tang, L. Jiang, B. Zou, J. Yang, H. Huang, Immobilization of

ED

Burkholderia cepacia lipase on functionalized ionic liquids modified mesoporous silica SBA-15, Process Biochem. 47 (2012) 2291-2299. [19] J. Yang, Y. Hu, L. Jiang, B. Zou, R. Jia, H. Huang, Enhancing the catalytic

PT

properties of porcine pancreatic lipase by immobilization on SBA-15 modified by functionalized ionic liquid, Biochem. Eng. J. 70 (2013) 46-54.

CC E

[20] B. Zou, Y. Hu, F. Cui, L. Jiang, D. Yu, H. Huang, Effect of surface modification of low cost mesoporous SiO2 carriers on the properties of immobilized lipase, J. Colloid Interf. Sci. 417 (2014) 210-216.

A

[21] Y. Mei, L. Miller, W. Gao, R.A. Gross, Imaging the distribution and secondary structure

of

immobilized

enzymes

using

infrared

microspectroscopy,

Biomacromolecules 4 (2003) 70-74. [22] H. Bao, Y. Pan, Y. Ping, N.G. Sahoo, T. Wu, L. Li, J. Li, L.H. Gan, Chitosan-functionalized graphene oxide as a nanocarrier for drug and gene delivery, Small 7 (2011) 1569-1578. 22

[23] K.M. Gregorio-Jauregui, M. Guadalupe Pineda, J.E. Rivera-Salinas, G. Hurtado, H. Saade, J.L. Martinez, A. Ilyina, R.G. Lopez, One-step method for preparation of magnetic nanoparticles coated with chitosan, J. Nanomater. (2012). [24] Z.H. Luan, J.A. Fournier, J.B. Wooten, D.E. Miser, Preparation and characterization of (3-aminopropyl) triethoxysilane-modified mesoporous SBA-15 silica molecular sieves, Micropor. Mesopor. Mat. 83 (2005) 150-158.

IP T

[25] K. sing, D.H. everett, R. haul, L. moscou, R.A. pierotti, J. rouquerol, T. siemieniewska, Reporting physisorption data for gas solid systems with special

SC R

reference to the determination of surface-area and porosity (recommendations 1984). Pure Appl. Chem. 1985, 57, 603-619..

[26] J.B. Pang, J.E. Hampsey, Q.Y. Hu, Z.W. Wu, V.T. John, Y.F. Lu, Mesoporous

U

silica with Ia3d cubic structure and good thermal stability, Chem. Commun. (2004)

N

682-683.

A

[27] J.D. Cabral, M. Roxburgh, Z. Shi, L. Liu, M. McConnell, G. Williams, N. Evans, L.R. Hanton, J. Simpson, S.C. Moratti, B.H. Robinson, P.J. Wormald, S. Robinson, physiochemical

characterization,

M

Synthesis,

and

biocompatibility

of

a

ED

chitosan/dextran-based hydrogel for postsurgical adhesion prevention, J. Mater. Sci.-Mater. M. 25 (2014) 2743-2756. [28] T. Zelinski, H. Waldmann, Cross-linked enzyme crystals (CLECs): Efficient and

PT

stable biocatalysts for preparative organic chemistry, Angew. Chem. Int. Edit. 36 (1997) 722-724.

CC E

[29] D. Yu, Y. Ma, S.J. Xue, L. Jiang, J. Shi, Characterization of immobilized phospholipase A(1) on magnetic nanoparticles for oil degumming application, LWT-Food Sci. Technol. 50 (2013) 519-525.

A

[30] L. Zang, J. Qiu, X. Wu, W. Zhang, E. Sakai, Y. Wei, Preparation of magnetic chitosan nanoparticles as support for cellulase immobilization, Ind. Eng. Chem. Res. 53 (2014) 3448-3454. [31] B. Zou, Y. Hu, D. Yu, J. Xia, S. Tang, W. Liu, H. Huang, Immobilization of porcine pancreatic lipase onto ionic liquid modified mesoporous silica SBA-15, Biochem. Eng. J. 53 (2010) 150-153. 23

[32] B. Zou, Y. Hu, D. Yu, L. Jiang, W. Liu, P. Song, Functionalized ionic liquid modified mesoporous silica SBA-15: A novel, designable and efficient carrier for porcine pancreas lipase, Colloid. Surface. B 88 (2011) 93-99. [33] Z. Chen, L. Liu, X. Wu, R. Yang, Synthesis of Fe3O4/P(St-AA) nanoparticles for enhancement of stability of the immobilized lipases, RSC Adv. 6 (2016) 108583-108589.

porcine

pancreatic

lipase

on

carboxyl-activated

magnetic

IP T

[34] Y. Zhu, X. Ren, Y. Liu, Y. Wei, L. Qing, X. Liao, Covalent immobilization of

nanoparticles:

SC R

Characterization and application for enzymatic inhibition assays, Mat. Sci. Eng. C-Mater. 38 (2014) 278-285.

[35] Y. Liu, C. Guo, C. Liu, Novel Magnetic cross-linked lipase aggregates for

U

improving the resolution of (R, S)-2-octanol, Chirality 27 (2015) 199-204.

N

[36] M. Seenuvasan, C.G. Malar, S. Preethi, N. Balaji, J. Iyyappan, M.A. Kumar, K.S.

A

Kumar, Immobilization of pectinase on co-precipitated magnetic nanoparticles for enhanced stability and activity, Res. J. Biotechnol. 8 (2013) 24-30. Kabsch,

C.

of

Dictionary

of

hydrogen-bonded

and

ED

structure-pattern-recognition

Sander,

M

[37] W.

A

CC E

PT

Biopolymers 22 (1983) 2577-2637.

24

protein geometrical

secondary features,

25

A ED

PT

CC E

IP T

SC R

U

N

A

M