Hydrophilic polyethylenimine modified magnetic graphene oxide composite as an efficient support for dextranase immobilization with improved stability and recyclable performance

Accepted Manuscript Title: Hydrophilic polyethylenimine modified magnetic graphene oxide composite as an efficient support for dextranase immobilization with improved stability and recyclable performance Authors: Yajie Wang, Qiang Wang, Xiaoping Song, Jingjing Cai PII: DOI: Reference:

S1369-703X(18)30380-2 https://doi.org/10.1016/j.bej.2018.10.015 BEJ 7066

To appear in:

Biochemical Engineering Journal

Received date: Revised date: Accepted date:

15-6-2018 13-9-2018 14-10-2018

Please cite this article as: Wang Y, Wang Q, Song X, Cai J, Hydrophilic polyethylenimine modified magnetic graphene oxide composite as an efficient support for dextranase immobilization with improved stability and recyclable performance, Biochemical Engineering Journal (2018), https://doi.org/10.1016/j.bej.2018.10.015 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.

Hydrophilic polyethylenimine modified magnetic graphene oxide composite as an efficient support for dextranase immobilization with improved stability and recyclable performance

IP T

Yajie Wang*, Qiang Wang, Xiaoping Song, and Jingjing Cai

Department of Pharmacy, Anhui Medical College, Hefei, 230601, Anhui, P. R. China

SC R

E-mail: [email protected];

Highlights

U

1. Successful immobilization of dextranase on magnetic graphene oxide composite

N

2. A loading capacity of 305.1 mg g-1 of immobilized dextranase was achieved

A

3. The immobilized dextranase possessed excellent reusability, even after 20 cycles

M

4. The unique structural merits ensure the improved stability and reusability

ED

ABSTRACT

Development of appropriate support for enzyme immobilization with approving loading capacity, stability and recyclable performance is a significant topic in enzyme

PT

chemistry. In this study, the hydrophilic polyethylenimine (PEI) modified magnetic graphene oxide (GO) composite (Fe3O4@PDA@GO-PEI) was fabricated and acted as

CC E

an efficient solid support for the immobilization of dextranase to improve the stability and recyclable performance. The GO nanosheet component not only provided abundant binding sites for decorating PEI molecules and further increasing the loading capacity

A

of immobilized dextranase, but also acted as a soft matrix for enhancing the stability of immobilized dextranase. In addition, in combination of the unique magnetic separation property, the recyclable ability of immobilized dextranase was further improved. The loading amount of immobilized dextranase on as-developed Fe3O4@PDA@GO-PEI was determined to be 305.1 mg g-1, which was 1.61 times that on the Fe3O4@PDA-PEI without GO component. Moreover, the as-developed Fe3O4@PDA@GO-PEI1

dextranase exhibited high immobilization efficiency (76.27 %) and enzymatic activity (83.67 %), improved stabilities in wide temperature conditions and pH solutions, and excellent reusability (85.78 % of its initial activity after 20 cycles). These experimental results suggest that the immobilized dextranase is promising for various applications in biocatalytic engineering fields.

IP T

Keywords: dextranase immobilization; polyethylenimine; graphene oxide; magnetic

SC R

particles; enzyme chemistry; biocatalyst

1. Introduction

Dextranase (1, 6-α-D-glucan-6-glucanohydrolase, EC 3.2.1.11) is an important

U

hydrolase, and it has received attracting noteworthy attentions in drug

N

formulation, vaccine, cosmetic, dental care, manufacturing blood substitute,

A

cancer antibody activator, oligosaccharide-synthesis, sugar-processing, and food-processing industries [1-9]. Recently, the dextranases with high enzyme

M

activities have been produced from several microorganisms [10-12], but some

ED

improvements are needed for further application. The enzyme activity was impressible in variable temperature and pH conditions. In addition, it was difficult to separate the dextranase from mixture solution, and the quality of

PT

product was influenced [13-14]. In this regard, the immobilization of dextranase in solid supports is an effective approach to improve its stability and reusability.

CC E

Up to now, several matrixes including chitosan hydrogel microsphere [15], porous glass [16], chitin, gelatin and bovine serum albumin [17], alginate bead [18-19], hydroxyapatite and bentonite [20], and Eupergit C [21] have been

A

employed for immobilizing dextranase. The improved immobilization yield and easy of recovery were realized, but the loading capacity, enzyme stability and reusability were still unsatisfied, especially for practical applications. Therefore, the development of appropriate immobilization supports for improving the comprehensive properties of immobilized dextranase is highly required.

2

In recent years, magnetic nanoparticles based on magnetite (Fe3O4) have received increasing attentions in biological and medical fields [22-23]. The unique character of this material is the magnetic responsive property, the magnetic composites can be facile and easy to be separated from the mixture solution using an external magnetic field, resulting in ease of manipulation, high separation efficiency, and good reusability [24-26]. A variety of functionalized

IP T

magnetic nanoparticles were fabricated for the immobilization of enzymes,

including trypsin [27-28], laccase [29], lipase [30-31], etc. Lately, the

SC R

polyethylenimine modified magnetic nanoparticles were synthesized for immobilizing dextranase, and the stability and reusability of immobilized

dextranase were slightly improved [32]. This research revealed that the

U

functionalized magnetic nanoparticles were promising for the immobilization of

N

dextranase. Additionally, the chemically covalent binding method showed higher

A

immobilization efficiency and immobilized activity than physical adsorption approach for immobilizing dextranase [33]. Besides, the glutaraldehyde coupling

M

process proved to be superior to several other covalent methods [16]. Moreover,

ED

the relatively low specific surface area and rigid surface of pristine magnetic nanoparticles may limits the loading capacity and catalytic activity of immobilized enzymes. In this issue, the integration of magnetic nanoparticles and

PT

other components which can offer large specific surface area may brings the relevant solutions.

CC E

Graphene oxide (GO) is a typical two-dimensional layer material with one-

atom thickness and various hydroxyl, epoxy, carbonyl and carboxyl groups on the surface, which provides a large specific surface area and abundant binding

A

sites

for

loading

decorations

[34-35].

These

unique

structural

and

physical/chemical properties making GO as a promising candidate in biological fields, such as drug delivery, cell imaging, and enzyme immobilization [35-39]. The protein enzymes can be directly conjugated with GO, but the limited amount of functional groups influences the loading capacities and accessibility of immobilized enzymes. In this regard, several intermediates including poly-lysine, 3

poly-glycidylmethacrylatel brushes and polyethylenimine with abundant groups were employed to remarkably increase the active sites [40-42]. Moreover, GO based magnetic nanocomposites have been prepared and proposed for the enzyme immobilization [43-46]. In the most commonly used solvothermal processes for preparing GO based magnetic nanocomposites, GO was reduced to a certain extent. The poor water dispersibility not only hindered the

IP T

immobilization efficiency of hydrophilic enzymes, but also caused the undesired non-specific adsorption. By contrast, the hydrophilic GO-based magnetic

SC R

nanocomposites were popular for the immobilization of hydrophilic enzymes.

In this study, the hydrophilic polyethylenimine modified magnetic graphene oxide composite (Fe3O4@PDA@GO-PEI) was synthesized and acted as an

U

efficient solid support for the immobilization of dextranase to improve the

N

stability and recyclable performance. Due to the flexible GO nanosheet

A

component, abundant amino groups, good hydrophilicity, and unique magnetic responsive ability of the nanocomposite, the as-developed Fe3O4@PDA@GO-

M

PEI-dextranase had a larger loading capacity than that of Fe 3O4@PDA-PEI-

ED

dextranase without GO component. In addition, the pH and thermal stabilities, and reusability of immobilized dextranase were investigated in detail. The experimental results demonstrated that the as-developed Fe3O4@PDA@GO-PEI

PT

is a promising support for improving the loading capacity, stability and

CC E

recyclable performance of immobilized dextranase.

2. Experimental section 2.1 Chemicals and materials

A

Graphene oxide (GO) nanosheet was supplied by Xianfeng Nanotech (Nanjing, China). Branched polyethylenimine (PEI, average molecular weight of 10 kDa), dopamine hydrochloride (DA), glutaraldehyde (GA, 50%), bovine serum albumin (BSA) and sodium borohydride (NaBH4) were purchased from SigmaAldrich (Shanghai, China). Dextran T70 (70 kDa) was obtained from Solarbio

4

(Beijing, China). Iron (Ⅲ) chloride hexahydrate (FeCl3·6H2O), sodium acetate (NaAc), ethanol, ethylene glycol (EG), and tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) were bought from Aladdin Industrial Corporation (Shanghai, China). Pure water (18.4 MΩ cm) used in all experiments was purified by a Milli-Q system (Millipore, Milford, MA, USA). All chemicals were

IP T

of analytical grade and used as received without further purification.

2.2 Preparation of polyethylenimine modified magnetic graphene oxide

SC R

(Fe3O4@PDA@GO-PEI)

Firstly, a solvothermal method was adopted for the synthesis of Fe3O4 nanoparticles [25]. Briefly, FeCl3·6H2O (2.7 g) and NaAc (7.2 g) were dissolved

U

in EG (75 mL) under magnetic stirring, respectively. The mixture was transferred

N

to a Teflon-lined stainless steel autoclave (100 mL), and kept at 200 oC for 8 h.

A

After naturally cooled to room temperature, the product of Fe3O4 was cleaned with ethanol and water for three times, respectively, and then dried at 60 oC

M

overnight.

Subsequently, Fe3O4 nanoparticles (400 mg) and DA (400 mg) were dispersed

ED

in Tris-HCl buffer (200 mL, 0.01 mol L-1) [47]. After mechanically stirred for 6 h at room temperature, the obtained product of Fe 3O4@PDA was magnetically

PT

separated and washed separately with ethanol and water for three times. Next, Fe3O4@PDA (400 mg) was dispersed in GO aqueous solution (400 mL,

CC E

1 mg mL-1), and mechanically stirred for 8 h, the product of Fe 3O4@PDA@GO was obtained after magnetically separated and cleaned with water for three times. Soon afterwards, Fe3O4@PDA@GO (100 mg) was dispersed in PEI aqueous

A

solution (100 mL, 10 mg mL-1) under mechanical stirring at room temperature. After executing the reaction for 12 h, Fe 3O4@PDA@GO-PEI was obtained.

2.3 Fabrication of dextranase functionalized polyethylenimine modified magnetic graphene oxide (Fe3O4@PDA@GO-PEI-dextranase)

5

The dextranase was produced from Paecilomyces lilacinus stain in the growth medium containing dextran T70 (1.0%), peptone (0.5%), K2HPO4·3H2O (0.4 %), MgSO4·7H2O (0.02 %), FeSO4·7H2O (0.001 %), and NaCl (0.05 %, w/v) with the pH value of 6.0 for 7 days at 30 oC. Subsequently, the culture broth was centrifuged at 10,000×g for 15 min at 4

o

C. The clear supernatant was

precipitated in an aqueous solution containing 80% (NH4)2SO4, and centrifuged

IP T

at 10,000×g for 15 min at 4 oC. The obtained precipitate was re-dissolved in sodium acetate buffer (0.1 mol L-1, pH 5.5) and then desalted using a Millipore

SC R

ultrafiltration membrane with a molecular weight cut off 10 kDa at 3,000×g for 30 min at 4 oC. The purified dextranase was stored in a refrigerator with a temperature of 4 oC.

U

The grafted PEI polymer on Fe3O4@PDA@GO-PEI was activated and

N

chemically bond with dextranase using GA as the cross-linking agent.

A

Fe3O4@PDA@GO-PEI (20 mg) was treated with GA in 20 mL of sodium phosphate buffer (0.1 mol L-1, pH 6.0) containing 5 % GA (w/v), and shook at

M

30 oC for 4 h. Then, the product was magnetically separated, washed with sodium phosphate buffer (0.1 mol L-1, pH 6.0) for three times, and then dispersed in 5

ED

mL of sodium phosphate buffer (0.1 mol L-1, pH 6.0) containing dextranase (2.0 mg mL-1) and NaBH4 (0.2 mg mL-1), After incubated at 30 oC for 4 h, the

PT

obtained Fe3O4@PDA@GO-PEI-dextranase was magnetically separated and washed with sodium phosphate buffer (0.1 mol L-1, pH 6.0) for three times. The

CC E

Fe3O4@PDA@GO-PEI-dextranase was stored at 4 oC for further use. For comparison, the Fe3O4@PDA-PEI without GO component was prepared

A

and adopted for the immobilization of dextranase.

2.4 Characterization and instruments Transmission electron microscopy (TEM) micrograph was obtained using a transmission electron microscope (Hitachi H-800, Japan). Fourier transform infrared (FTIR) spectroscopy was recorded with a FTIR-7600 spectrometer (Lambda Scientific, Australia). Zeta (ζ) potential measurement was carried out 6

on a zeta-potential analyser at 25

o

C in pure water (pH 7.0) (ZetaPlus,

Brookhaven Instrument Corporation). The Brunauer-Emmett-Teller (BET) specific surface area and Barrett-Joyner-Halenda (BJH) pore-size distribution were measured with a surface area and pore size analyzer (V-sorb 2800P, Gold APP instruments). Saturation magnetization curves were determined on a Physical Property Measurement System 9T (Quantum Design, San Diego, CA)

IP T

at room temperature. The UV-vis spectrum absorbance value was measured

SC R

using a Shimadzu UV-1700 spectrophotometer (Duisburg, Germany).

2.5 Determination of the enzymatic property of Fe3O4@PDA@GO-PEIdextranase

U

For determining the loading capacity of immobilized dextranase, the supernatant

N

solution and washing solutions in the preparation process of immobilized

A

dextranase were collected. The concentrations of residual dextranase were measured according to the Bradford protein assay using BSA as a standard

M

protein. The protein loading capacity was calculated as the mass ratio of

ED

immobilized dextranase to the solid support (mg g -1). Additionally, the immobilization efficiency was measured as the mass ratio of immobilized dextranase to the total purified dextranase (%).

PT

The dextranase activity was measured by executing the hydrolysis reaction of dextran T70 (2 %, w/v) in 1.6 mL of sodium acetate buffer (0.1 mol L-1, pH 5.5)

CC E

with free or immobilized dextranase solution (0.4 mL) for 15 min at 50 oC. The amount of reducing sugar was quantitatively measured using the 3,5dinitrosalicylic acid method [48-49]. One unit of dextranase activity is appointed

A

as the quality of dextranase that produces 1 μmol of reducing sugar (measured as maltose) in one minute. Absorbance at 540 nm was determined using a UV spectrophotometer. The influence of pH on dextranase activity was measured in two buffers with different pH values (sodium acetate buffer, 0.1 mol L-1, pH 3.0―5.5; sodium phosphate buffer, 0.1 mol L-1, pH 6.0―8.0). The free or immobilized dextranase 7

was incubated in the buffers with different pH values at 50 oC for 1 h. The relative activity is shown as a percentage of the maximum activity (100 %). The effect of temperature on dextranase activity was examined by incubating the free or immobilized dextranase in sodium acetate buffer (0.1 mol L-1, pH 5.5) at different temperature (30 to 70 oC) for 2 h. The relative activity is shown as a percentage of the maximum activity (100 %).

IP T

The thermal stabilities of free and immobilized dextranase were further studied

by incubating the enzyme at 60 oC for 10 h. In each one hour, the remaining

SC R

activity was measured in the same condition as previously described. The relative activity is presented as a percentage of initial activity (100 %).

The initial velocity for determining the kinetic constant of free or immobilized

U

dextranase was studied at different concentrations of dextran T70 varying from

N

1 to 50 mg mL-1 in sodium acetate buffer (0.1 mol L-1, pH 5.5). The kinetic values

A

of enzyme (Michaelis-Menten constant Km, and maximum reaction velocity Vmax) were calculated by a Lineweaver-Burk plots method.

M

The reusability of immobilized dextranase on Fe3O4@PDA@GO-PEI-

ED

dextranase was investigated by reusing the material after each cycle. In a whole cycle, the Fe3O4@PDA@GO-PEI-dextranase was dispersed in 2.0 mL of sodium acetate buffer (0.1 mol L-1, pH 5.5) containing dextran T70 (20 g L-1). After the

PT

hydrolysis reaction was performed at 50

o

C for 15 min, the collected

Fe3O4@PDA@GO-PEI-dextranase was cleaned with sodium acetate buffer (0.1

CC E

mol L-1, pH 5.5) for two times. The remaining activity is presented as a percentage of the initial activity (100 %). Three parallel measurements were performed in all tests in this section, and

A

the mean value was presented.

3. Results and discussion 3.1 Preparation and characterization of Fe3O4@PDA@GO-PEI-dextranase nanocomposite

8

The preparation process of Fe3O4@PDA@GO-PEI-dextranase nanocomposite was shown in Fig. 1. Fe3O4 nanoparticles were coated with a polydopamine (PDA) layer at first, and then GO nanosheet was directly encapsulated on the surface of Fe3O4@PDA through the strong π-π stacking and hydrogen bonding interactions between PDA and GO. Secondly, the branched PEI polymer was self-assembled to the surface of GO by electrostatic interactions. Finally,

M

A

N

U

SC R

to obtain Fe3O4@PDA@GO-PEI-dextranase nanocomposite.

IP T

dextranase was covalently bound to PEI by using GA as the cross-linking agent

PEI-dextranase.

ED

Fig. 1 Schematic illustration of the fabrication process of Fe 3O4@PDA@GO-

PT

The structures and morphologies of as-prepared materials were characterized by TEM. As displayed in Fig. 2a, the synthesized Fe3O4 nanoparticles were

CC E

spherical in shape, and the average diameter was about 90 nm. After coated with a PDA layer, a core-shell structure was formed with the shell thickness of approximately 20 nm (Fig. 2b). In addition, the GO nanosheets with crinkled

A

structure (Fig. 2c) were obviously observed on the surface of Fe3O4@PDA nanoparticles (Fig. 2d), revealing that the Fe3O4@PDA were tightly encapsulated by the rough and flexible GO nanosheets. Moreover, because of the relatively small size of Fe3O4@PDA nanoparticles and the relatively large size of GO nanosheets, several nanoparticles were encapsulated simultaneously by a spot of GO. More interestingly, the edge of GO nanosheets were spread out over 9

Fe3O4@PDA

nanoparticles

with

a

carpet-like

structure

without

any

agglomeration (Fig. 2d). The unique structure could remarkably expands the surface area, and it provides abundant accessible sites for PEI immobilization. Furthermore, the morphologies of as-prepared Fe3O4@PDA@GO-PEI and Fe3O4@PDA@GO-PEI-dextranase have slight changed (Fig. 2e, 2f), these results contributed to the gentle reaction processes for the self-assembly of PEI

CC E

PT

ED

M

A

N

U

SC R

IP T

and chemically covalent bonding of dextranase.

Fig.

2

TEM

images

of

(a)

Fe3O4,

(b)Fe3O4@PDA,

(c)

GO,

(d)

Fe3O4@PDA@GO, (e) Fe3O4@PDA@GO-PEI, and (f) Fe3O4@PDA@GO-PEI-

A

dextranase.

FTIR spectroscopy was adopted to examine the chemical structures of asprepared Fe3O4, Fe3O4@PDA, Fe3O4@PDA@GO, Fe3O4@PDA-GO-PEI, Fe3O4@PDA-GO-PEI-dextranase as well as pure PEI and purified dextranase. The characteristic absorption peak at 588 cm-1 corresponded to the stretching 10

vibration of Fe-O band of Fe3O4 (Fig. 3a). The absorption peaks observed at 1506 and 1606 cm-1 ascribed to the aromatic ring C-C vibration, and the absorption peak appeared at 1276 cm-1 corresponded to the phenolic C-OH stretching vibration of PDA (Fig. 3b), demonstrating the successful formation of Fe3O4@PDA. Additionally, two new absorption peaks appeared at 1051 and 1727 cm-1 can be attributed to the C-O stretching vibration and C=O stretching

IP T

vibration, respectively, revealing the successful introduction of GO (Fig. 3c). Besides, the pure PEI molecules showed a high-intensity absorption band at 1577

SC R

cm-1 (Fig. 3d), corresponding to the symmetric bending vibration of NH2 groups. While a low-intensity absorption band at the same peak in the FTIR spectrum of Fe3O4@PDA-GO-PEI was observed. Moreover, as shown in Fig. 3d and 3e, the

U

assembly of PEI molecules was also proved by the existence of the characteristic

N

absorption peaks at 2935 and 2852 cm-1, ascribing to the C-H stretching vibration

A

of CH2 groups connecting with the NH2 groups [50]. Furthermore, two absorption peaks appeared at 1646 and 1540 cm-1 in the FTIR spectra of purified

M

dextranase and Fe3O4@PDA-GO-PEI-dextranase corresponded to amide I and

ED

amide II bands of enzyme protein molecules (Fig. 3f and 3g). These results demonstrate the successful immobilization of dextranase on the as-developed

A

CC E

PT

solid support.

11

IP T SC R U N A M ED PT

Fig. 3 FTIR spectra of (a) Fe3O4, (b) Fe3O4@PDA, (c) Fe3O4@PDA@GO, (d)

CC E

pure PEI, (e) Fe3O4@PDA@GO-PEI, (f) purified dextranase, and (g)

A

Fe3O4@PDA@GO-PEI-dextranase.

Zeta (ζ) potential measurement was employed to check the surface charges of

Fe3O4,

Fe3O4@PDA,

Fe3O4@PDA@GO,

Fe3O4@PDA@GO-PEI,

and

Fe3O4@PDA@GO-PEI-dextranase. As presented in Fig. 4a, the Fe3O4 nanoparticles exhibited a negative zeta potential of -18.87 mV, while the potential of Fe3O4@PDA declined to -25.3 mV after the surface coating of a PDA layer (Fig. 4b). Moreover, because of the existing hydroxyl and carboxyl groups 12

on GO surface, the potential of Fe3O4@PDA@GO further decreased to -39.78 mV (Fig. 4c). Importantly, the potentials of Fe3O4@PDA@GO-PEI and Fe3O4@PDA@GO-PEI-dextranase dramatically changed to the positive values of + 35.8 mV and +10.68 mV, respectively, as shown in Fig. 4d and 4e, showing the assembly of PEI molecules and the chemical bonding of dextranase in

A

N

U

SC R

IP T

sequence.

M

Fig. 4 Zeta potentials of (a) Fe3O4, (b)Fe3O4@PDA, (c) Fe3O4@PDA@GO, (d)

A

CC E

PT

ED

Fe3O4@PDA@GO-PEI, and (e) Fe3O4@PDA@GO-PEI-dextranase.

13

IP T SC R U N A M ED

Fig. 5 (a) Nitrogen adsorption-desorption isotherms and (b) BJH desorption pore

CC E

dextranase.

PT

size distribution curves of Fe3O4@PDA@GO-PEI and Fe3O4@PDA@GO-PEI-

The Fe3O4@PDA@GO-PEI and Fe3O4@PDA@GO-PEI-dextranase were

characterized by the Brunauer-Emmett-Teller (BET) specific surface area and

A

Barrett-Joyner-Halenda (BJH) pore size analyses. As shown in Fig. 5a, the BET specific surface area of Fe3O4@PDA@GO-PEI and Fe3O4@PDA@GO-PEIdextranase were measured to 54.2 and 49.2 m2 g-1, respectively. The relatively low values may ascribed to the agglomeration of as-developed nanocomposite in drying approaches. In addition, the Fe3O4@PDA@GO-PEI displayed a pore volume of 0.62 cm3 g-1 and several pore sizes at 4.0, 53.2, 94.6, and 155.0 nm 14

(Fig. 5b). The multiple pore size distribution was resulted from the assembly of Fe3O4 nanoparticles and GO nanosheets. In contrast, the smaller pore volume (0.34 cm3 g-1) and pore sizes (3.7, 80.9, and 152.7 nm) were observed for Fe3O4@PDA@GO-PEI-dextranase. The reductive pore volume and pore size can be attributed to the immobilization of enzyme protein (i.e. dextranase) on Fe3O4@PDA@GO-PEI. From these results, it can be speculated that the

IP T

dextranase may immobilized on the internal surface of Fe3O4@PDA@GO-PEI. Furthermore, it also demonstrates the successful immobilization of dextranase on

SC R

Fe3O4@PDA@GO-PEI.

The unique magnetic responsive properties of as-prepared Fe3O4 and Fe3O4@PDA@GO-PEI-dextranase were characterized by a vibrating sample

U

magnetometer. The magnetic hysteresis curves in Fig. 6 revealed that the as-

remanence

and

coercivity,

which

demonstrated

their

A

hysteresis,

N

prepared Fe3O4 and Fe3O4@PDA@GO-PEI-dextranase have no obvious

superparmagnetic behaviours. The saturation magnetization value of obtained

M

Fe3O4@PDA@GO-PEI-dextranase was determined to 31.57 emu g-1. The strong

A

CC E

PT

ED

magnetization was sufficient for magnetic separation from an aqueous solution.

Fig. 6 Magnetization curves of (a) Fe3O4, and (b) Fe3O4@PDA@GO-PEIdextranase. 15

Furthermore, as shown in Fig. 7a, the Fe3O4@PDA@GO-PEI-dextranase nanocomposite was well-dispersed in sodium acetate buffer (0.1 mol L-1, pH 5.5), showing the hydrophilic character. Additionally, the Fe 3O4@PDA@GO-PEIdextranase particles can be rapidly separated from the solution when an external magnet was applied on the outside wall of the vessel (Fig. 7b). Moreover, as

IP T

displayed in Fig. 7c, after gentle shaking, the hydrophilic Fe 3O4@PDA@GO-

PEI-dextranase particles were re-dispersed in sodium acetate buffer (0.1 mol L, pH 5.5).

M

A

N

U

SC R

1

Fig. 7 Digital images of (a) the well-dispersed Fe3O4@PDA@GO-PEI-dextranase

ED

suspension solution in sodium acetate buffer (0.1 mol L-1, pH 5.5), (b) the magnetic separation under an external magnet (arrows indicate the magnetic nanocomposites

PT

adsorbed on the internal surface of the glass vessel next to the magnet), and (c) the redispersion of Fe3O4@PDA@GO-PEI-dextranase suspension solution after gentle

CC E

shaking.

3.2 Loading capacity and enzymatic activity of immobilized dextranase

A

For the sake of achieving the maximum loading capacity and enzymatic activity of immobilized dextranase. The effects of two main factors including initial dextranase concentration and reaction time were investigated. Simultaneously, the immobilization support of Fe3O4@PDA-PEI without GO component was fabricated and employed for the chemical bonding of dextranase as a control group. As shown in Fig. 8a and 8b, the loading capacities of immobilized 16

dextranase on Fe3O4@PDA@GO-PEI and Fe3O4@PDA-PEI were increased with incremental initial dextranase concentration and reaction time until reaching the plateaus. In consideration of the immobilization efficiency of dextranase (Fig. 8c), the loading capacity of immobilized dextranase on Fe 3O4@PDA@GO-PEI was selected to 305.1 mg g-1 when the concentration of dextranase at 2.0 mg mL1

and the reaction time of 6 h. In this condition, the immobilized efficiency was

IP T

calculated to 76.27 %. Similarly, when the concentration of dextranase at 2.0 mg mL-1, the loading capacity of immobilized dextranase on Fe 3O4@PDA-PEI was

dextranase on

SC R

measured to 189.3 mg g-1. The loading capacity of immobilized

Fe3O4@PDA@GO-PEI was 1.61 times that of Fe3O4@PDA-PEI, the result may attributed to the large surface area and abundant accessible reaction sites on the

U

flexible GO nanosheets for the subsequent self-assembly of PEI molecules and

N

chemical bounding of dextranase.

A

The effects of initial dextranase concentration and reaction time on the enzymatic activities of immobilized dextranase were investigated and the results

M

were displayed in Fig. 8c, 8d, Fig. S1, and S2 in Supplementary material. With

ED

increasing the initial dextranase concentration, the enzymatic activities of Fe3O4@PDA@GO-PEI-dextranase and Fe3O4@PDA-PEI-dextranase raised gradually, but suddenly declined in the relatively high concentrations of

PT

dextranase (Fig. 8c). The decreased enzymatic activity may due to the excessive dextranase loading on the surface and the limited mass-transfer effect caused by

CC E

the adjacent dextranases, which retarded the diffusion of the reactants and products. The maximum enzymatic activities of Fe 3O4@PDA@GO-PEIdextranase and Fe3O4@PDA-PEI-dextranase were realized in the initial

A

dextranase concentration of 2.0 and 1.5 mg mL -1, respectively (Fig. 8c, Fig. S1, Supplementary

material).

Analogously,

Fe3O4@PDA@GO-PEI-dextranase

and

the

enzymatic

activities

Fe3O4@PDA-PEI-dextranase

of were

increased with performing the reaction processes. But further extending the reaction time, the enzymatic activities decreased. The phenomenon was similar to the previous results [15, 32]. The result may attributed to excessive cross17

linking reaction between multiple GA and a dextranase. The maximum enzymatic activities of Fe3O4@PDA@GO-PEI-dextranase and Fe3O4@PDAPEI-dextranase were achieved at the reaction time of 6 h and 4 h, respectively (Fig. 8d, Fig. S2, Supplementary material). As a result, the enzymatic activity of Fe3O4@PDA@GO-PEI-dextranase was determined to be 83.67 % compared to

PT

ED

M

A

N

U

SC R

IP T

that of free dextranase.

CC E

Fig. 8 Effects of initial dextranase concentration (a) and reaction time (b) on the loading capacities of immobilized dextranase on Fe3O4@PDA@GO-PEI and Fe3O4@PDAPEI; Effects of initial dextranase concentration (c) and reaction time (d) on the

A

immobilization efficiencies and relative enzyme activities of the Fe3O4@PDA@GOPEI-dextranase.

3.3 pH and temperature stabilities of free and immobilized dextranase The effect of pH values on the relative activities of free and immobilized dextranase were studied in the pH range of 3―8. As revealed in Fig. 9, the 18

maximum enzyme activities of free dextranase, Fe3O4@PDA-PEI-dextranase, and Fe3O4@PDA@GO-PEI-dextranase were observed at pH 5.5, 6, and 6, respectively, indicating that the pH optimum of immobilized dextranase shifted into a more alkaline solution. More importantly, the immobilized dextranase on Fe3O4@PDA-PEI

and

Fe3O4@PDA@GO-PEI

exhibited

higher

relative

activities both in acidic and alkaline pH ranges when compared to its free form.

IP T

For example, the Fe3O4@PDA-PEI-dextranase and Fe3O4@PDA@GO-PEIdextranase retained 56.26 % and 66.36 % of the initial activities at pH 3,

SC R

respectively, which were higher than free dextranase at the same pH (34.13 %). Moreover, the higher enzymatic activity of immobilized dextranase on

Fe3O4@PDA@GO-PEI than that on the Fe3O4@PDA-PEI might attributed to the

U

highly flexible GO nanosheets on the immobilization support and improved

A

CC E

PT

ED

M

A

adaptability to acid and alkali conditions.

N

tolerability to the pH variance. In short, the immobilized dextranase showed good

Fig. 9 Effect of pH value on the enzymatic activities of free dextranase, Fe3O4@PDA-PEI-dextranase, and Fe3O4@PDA@ GO-PEI-dextranase.

Subsequently, the effect of temperature on the relative activities of free and immobilized dextranase were shown in Fig. 10. The optimum temperature for 19

free dextranase was 50 oC. While those of Fe3O4@PDA-PEI-dextranase and Fe3O4@PDA@GO-PEI-dextranase were observed at the same temperature of 55 o

C. The shifts toward higher temperature for the immobilized dextranase were

related to fixed dextranase causing an increased activation energy for enzyme for binding the substrate. Besides, the immobilized dextranases presented the broader temperature profiles compared with the free dextranase. For instance, the

IP T

free dextranase maintained more than 80 % of its initial activity in the range

between about 45 and 60 oC, while the ranges increased to 45―70 oC for both and

Fe3O4@PDA@GO-PEI-dextranase.

The

SC R

Fe3O4@PDA-PEI-dextranase

improved resistance against temperature may attributed to conformational

CC E

PT

ED

M

A

N

U

rigidity of dextranase after immobilization.

Fig. 10 Effects of temperature on the enzymatic activities of free dextranase,

A

Fe3O4@PDA-PEI-dextranase, and Fe3O4@ PDA@GO-PEI-dextranase.

The thermal stabilities were further investigated with free dextranase and

Fe3O4@PDA@GO-PEI-dextranase at 60 oC. As shown in Fig. 11, the relative activity of free dextranase decreased much rapider than Fe 3O4@PDA@GO-PEIdextranase with the increasing of incubating time. After incubation at 60 oC for 5 h, the free dextranase only remained 57.36 % of its initial activity. While 20

Fe3O4@PDA@GO-PEI-dextranase still remained 91.69 % of its initial activity. Moreover, the relative activity of Fe3O4@PDA@GO-PEI-dextranase was over 80 % after the incubation at 60 oC for 10 h. The improved thermal stability of

A

N

U

SC R

IP T

immobilized dextranase favors long-term use in practical applications.

M

Fig. 11 Thermal stabilities of free dextranase and Fe3O4@PDA@GO-PEI-dextranase

ED

at 60 oC.

3.4 Kinetic parameters of free and immobilized dextranase

PT

Kinetic parameters of free and immobilized dextranase were studied. According to a liner transformation of the Lineweaver-Burk plot method [51], the

CC E

Michaelis-Menten constant (Km) values of free dextranase, Fe3O4@PDA@GOPEI-dextranase and Fe3O4@PDA-PEI-dextranase were calculated to be 9.32 ± 0.45, 10.95 ± 1.32, and 10.68 ± 0.85 mmol L-1, respectively. While the maximum

A

velocity (Vmax) of Fe3O4@PDA@GO-PEI-dextranase (124.6 ± 1.1 µmol min-1) and Fe3O4@PDA-PEI-dextranase (130.2 ± 1.7 µmol min-1) were lower than that of free dextranase (146.5 ± 1.8 µmol min-1). The slight raise of Km may be ascribed to the limited accessibility of substrates to the active sites of immobilized enzymes, resulting in lower affinity between immobilized dextranase and substrates. The similar results have been observed for the 21

immobilization of dextranase of Aspergillus subolivaceus [17], Penicillium lilacinum [21], and Chaetomium erraticum [52]. On the other hand, the immobilization of enzyme on solid support can lead to a decrease in catalytic activity. Hence, the Vmax of immobilized dextranase was lower than that of free dextranase. Moreover, an increase of Km and decrease of Vmax for other enzymes

IP T

after immobilization have also been reported in the researches [53-55].

3.5 Reusability of immobilized dextranase

SC R

The reusability of immobilized enzyme is an important parameter for potential

industrial applications. For investigating the reusability of Fe3O4@PDA@GOPEI-dextranase, the nanocomposite was repeated use for 20 cycles. As presented

U

in Fig. 12, the relative activity of Fe3O4@PDA@GO-PEI-dextranase was slowly

N

decreased, and it maintained 85.78 % of the initial activity after 20th cycles. The

hydrophilic

A

reduction of enzymatic activity may ascribed to the not full recovery of Fe3O4@PDA@GO-PEI-dextranase

nanocomposite

and

the

M

inactivation of immobilized dextranase in the operation processes. It could be

ED

summarized a conclusion that the as-prepared Fe3O4@PDA@GO-PEI-

A

CC E

PT

dextranase exhibited good stability and operability.

Fig. 12 Reusability of as-prepared Fe3O4@PDA@GO-PEI-dextranase. 22

3.6 Comparison with various solid supports In the current research, dextranase was covalently immobilized on asprepared Fe3O4@PDA@GO-PEI. Several main properties of immobilized dextranase were compared with some of previous researches (Table 1). The pH tolerance feature of immobilized dextranase on Fe 3O4@PDA@GO-PEI was

IP T

better than several immobilized supports. For example, the immobilized

dextranase on Fe3O4@PDA@GO-PEI retained above 80 % of the initial

SC R

activities at pH range from 4.0 to 8.0, which was higher than that on chitosan hydrogel microspheres (from about 6.0 to 8.5) [15] and bovine serum albumin (from about 5.5 to 8.0) [17]. Moreover, the temperature tolerance property of

U

immobilized dextranase on Fe3O4@PDA@GO-PEI was lower than several solid

N

supports [15, 17, 21]. This phenomenon may be ascribed to the relatively low

A

temperature tolerance of initial dextranase. On the other hand, the loading capacity of immobilized dextranase on Fe3O4@PDA@GO-PEI was obviously

M

higher than other reported solid supports [15, 17, 32-33]. This result can be

ED

attributed to the nano-sized Fe3O4 and GO materials, and abundant covalent bonding sites of the as-developed nanocomposites. The high loading capacity of immobilized enzymes on the nanomaterials have been reported in previous

PT

reports [31, 40-46]. In addition, the recycling performance of immobilized dextranase on Fe3O4@PDA@GO-PEI was superior to several solid supports,

CC E

including chitosan hydrogel microspheres [15], bovine serum albumin [17], Fe3O4@SiO2-PEI [32], and chitosan [33]. The improved reusability may attributed to the multiple component and unique structure of as-developed

A

immobilization support in this research. In combination of these advantages of high enzyme activity, good stability and reusability, and unique magnetic separation feature, the as-developed dextranase-immobilized nanocomposite has great potential in biocatalytic engineering fields, such as oligosaccharidesynthesis, vaccine-processing, and sugar-processing industries.

23

Table 1 Comparison of properties of immobilized dextranase on various solid supports Immobilization

Support

Loading

Reusability

capability

Ref.

pH

Temperature

(mg g-1)

Time

Activity (%)

Covalent binding

7.5

60 oC

9.0 a

14

40

[15]

Bovine serum albumin

Covalent binding

6.0

50 oC

0.0066 a

5

76

[17]

Eupergit C

Covalent binding

5.3

35 oC

―

―

―

[21]

Fe3O4@SiO2-PEI

Covalent binding

5.5

60 oC

217.2

15

IP T

technique

Optimum conditions

83.26

[32]

Chitosan

Covalent binding

8.0

80 oC

8.6 a

12

81

[33]

Fe3O4@PDA@GO-PEI

Covalent binding

6.0

55 oC

305.1

20

85.78

Chitosan hydrogel

The loading capability was calculated according to the reaction condition and

U

a

SC R

microspheres

A

N

immobilization efficiency in the corresponding research.

M

4. Conclusion

In this study, the hydrophilic polyethylenimine modified magnetic graphene

ED

oxide nanocomposite was prepared and acted as an efficient solid support for immobilizing dextranase to improve the stability and recyclable performance.

PT

The graphene oxide nanosheets not only provided abundant sites for the assembly of polyethylenimine molecules for increasing the loading capacity of

CC E

immobilized dextranase, but also acted as a soft matrix for enhancing the stability of immobilized dextranase. Benefit from the multiple component and unique structure of immobilization support, the immobilized dextranase exhibited

A

excellent properties including high loading capacity and immobilization efficiency, high pH and temperature endurances, and good reusability. It is believed that the dextranase-immobilized magnetic composite is promising in biocatalytic engineering fields.

Acknowledgements 24

This work

This study was supported by the Key Programs of Anhui Provincial Natural Science Research in Colleges and Universities (No. KJ2016A378, No. KJ2017A695), the Programs of Anhui Provincial Natural Science Research in Colleges and Universities (No. 12925KJ2015B02), and the Anhui Natural Science Foundation (No. 1808085MC86).

IP T

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in the online

SC R

version, at http://dx/doi.org/10.1016/j.bej.2018xxxxx.

References

U

[1] E. Khalikova, P. Susi, T. Korpela, Microbial dextran-hydrolyzing enzymes:

N

Fundamentals and applications. Microbiol. Mol. Biol. Rev. 69 (2005) 306-325.

A

[2] X. Wang, M. Lu, S. Wang, Y. Fang, D. Wang, W. Ren, G. Zhao, The atmospheric and room-temperature plasma (ARTP) method on the dextranase activity and structure.

M

Int. J. Biol. Macromol. 70 (2014) 284-291.

ED

[3] Y.-L. Jiao, S.-J. Wang, M.-S. Lv, B.-H. Jiao, W.-J. Li, Y.-W. Fang, S. Liu, Characterization of a marine-derived dextranase and its application to the prevention of dental caries. J. Ind. Microbiol. Biot. 41 (2014) 17-26.

PT

[4] W. Gan, H. Zhang, Y. Zhang, X. Hu, Biosynthesis of oligodextrans with different M-w by synergistic catalysis of dextransucrase and dextranase. Carbohyd. Polym. 112

CC E

(2014) 387-395.

[5] Y.-Q. Zhang, R.-H. Li, H.-B. Zhang, M. Wu, X.-Q. Hu, Purification, characterization, and application of a thermostable dextranase from Talaromyces

A

pinophilus. J. Ind. Microbiol. Biot. 44 (2017) 317-327. [6] S. Bhatia, G. Bhakri, M. Arora, S. K. Uppal, S. K. Batta, Dextranase production from Paecilomyces lilacinus and its application for dextran removal from sugarcane juice. Sugar Tech. 12 (2010) 133-138. [7] A. K. Goulas, D. A. Fisher, G. K. Grimble, A. S. Grandison, R. A. Rastall, Synthesis of isomaltooligosaccharides and oligodextrans by the combined use of dextransucrase 25

and dextranase. Enzyme Microb. Tech. 35 (2004) 327-338. [8] G. Eggleston, A. Monge, Optimization of sugarcane factory application of commercial dextranases. Process Biochem. 40 (2005) 1881-1894. [9] M. Bashari, F. Tounkara, M. H. Abdelhai, C. Lagnika, X. M. Xu, Z. Y. Jin, Impact of dextranase on sugar manufacturing and its kinetic on the molecular weights of remaining dextran. Sugar Tech. 15 (2013) 84-93.

IP T

[10] J.-J. Wang, Z.-Y. Wang, X.-P. He, B.-R. Z hang, Integrated expression of the alphaamylase, dextranase and glutathione gene in an industrial brewer's yeast strain. World

SC R

J. Microbio. Biot. 28 (2012) 223-231.

[11] P. Rerngsamran, P. Temjitpukdee, N. Assavasirijinda, S. Chareonpornwattana, S. Thaniyavarn, Cloning, characterization, and heterologous expression of a dextranase

U

gene from Penicillium pinophilum SMCU3-14. Scienceasia 40 (2014) 405-413.

N

[12] R. R. Zohra, A. Aman, A. Ansari, M. S. Haider, S. A. Ul Qader, Purification,

A

characterization and end product analysis of dextran degrading endodextranase from Bacillus licheniformis KIBGE-IB25. Int J Biol Macromo. 78 (2015) 243-248.

M

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

ED

how. Chem. Soc. Rev. 42 (2013) 6223-6235. [14] S. Chalane, C. Delattre, P. Michaud, A. Lebert, C. Gardarin, D. Kothari, C. Creuly, A. Goyal, A. Strancar, G. Pierre, Optimized endodextranase-epoxy CIM (R) disk

PT

reactor for the continuous production of molecular weight-controlled prebiotic isomalto-oligosaccharides. Process Biochem. 58 (2017) 105-113.

CC E

[15] F. Shahid, A. Aman, M. A. Nawaz, A. Karim, S. A. Ul Qader, Chitosan hydrogel microspheres: an effective covalent matrix for crosslinking of soluble dextranase to increase stability and recycling efficiency. Bioproc. Biosyst. Eng. 40 (2017) 451-461.

A

[16] J. Rogalski, J. Szczodrak, M. Pleszczynska, J. Fiedurek, Immobilisation and kinetics of Penicillium notatum dextranase on controlled porous glass. J. Mol. Catal. BEnzym. 3 (1997) 271-283. [17] A. B. El-Tanash, E. El-Baz, A. A. Sherief, Properties of Aspergillus subolivaceus free and immobilized dextranase. Eur. Food Res. Technol. 233 (2011) 735-742. [18] Z. Olcer, A. Tanriseven, Co-immobilization of dextransucrase and dextranase in 26

alginate. Process Biochem. 45 (2010) 1645-1651. [19] J. M. R. T ingirikari, W. F. Gomes, S. Rodrigues, Efficient Production of Prebiotic Gluco-oligosaccharides in Orange Juice Using Immobilized and Co-immobilized Dextransucrase. App. Biochem. Biotech. 183 (2017) 1265-1281. [20] F. A. Erhardt, H.-J. Joerdening, Immobilization of dextranase from Chaetomium erraticum. J. Biotechnol. 131 (2007) 440-447.

IP T

[21] Y. Aslan, A. Tanriseven, Immobilization of Penicillium lilacinum dextranase to produce isomaltooligosaccharides from dextran. Biochem. Eng. J. 34 (2007) 8-12.

SC R

[22] Y. Hu, S. Mignani, J.-P. Majoral, M. Shen, X. Shi, Construction of iron oxide

nanoparticle-based hybrid platforms for tumor imaging and therapy. Chem. Soc. Rev. 47 (2018) 1874-1900.

U

[23] Z. Liu, Y. Liu, S. Shen, D. Wu, Progress of recyclable magnetic particles for

N

biomedical applications. J. Mater. Chem. B 6 (2018) 366-380.

A

[24] K. Zhu, Y. Ju, J. Xu, Z. Yang, S. Gao, Y. Hou, Magnetic Nanomaterials: Chemical Design, Synthesis, and Potential Applications. Acc. Chem. Res. 51 (2018) 404-413.

M

[25] Z. Xiong, Y. Chen, L. Zhang, J. Ren, Q. Zhang, M. Ye, W. Zhang, H. Zou, Facile

ED

Synthesis of Guanidyl-Functionalized Magnetic Polymer Microspheres for Tunable and Specific Capture of Global Phosphopeptides or Only Multiphosphopeptides. ACS Appl. Mater. Interfaces 6 (2014) 22743-22750.

PT

[26] Z. Xiong, Y. Ji, C. Fang, Q. Zhang, L. Zhang, M. Ye, W. Zhang, H. Zou, Facile Preparation of Core-Shell Magnetic Metal-Organic Framework Nanospheres for the

CC E

Selective Enrichment of Endogenous Peptides. Chem. Eur. J. 20 (2014) 7389-7395. [27] L. Zhang, B. Wang, S. Wang, W. Zhang, Recyclable trypsin immobilized magnetic nanoparticles based on hydrophilic polyethylenimine modification and their proteolytic

A

characteristics. Anal. Meth. 10 (2018) 459-466. [28] G. Cheng, P. Chen, Z.-G. Wang, X.-J. Sui, J.-L. Zhang, J.-Z. Ni, Immobilization of trypsin onto multifunctional meso-fmacroporous core-shell microspheres: A new platform for rapid enzymatic digestion. Anal. Chim. Acta 812 (2014) 65-73. [29] T.-T. Xia, W. Lin, C.-Z. Liu, C. Guo, Improving catalytic activity of laccase immobilized on the branched polymer chains of magnetic nanoparticles under 27

alternating magnetic field. J. Chem. Technol. Biot. 93 (2018), 88-93. [30] Z. Chen, L. Liu, X. Wu, R. Y ang, Synthesis of Fe3O4/P(St-AA) nanoparticles for enhancement of stability of the immobilized lipases. RSC Adv. 6 (2016) 108583108589. [31] Chen, Z.; Liu, L.; Yang, R., Improved performance of immobilized lipase by interfacial activation on Fe3O4@PVBC nanoparticles. RSC Adv. 7 (2017) 35169-35174.

IP T

[32] Y. J. Wang, Q. Wang, X. P. Song, J. J. Cai, Improving the stability and reusability of dextranase by immobilization on polyethylenimine modified magnetic particles.

SC R

New J. Chem. 42 (2014) 8391-8399.

[33] M. A. Abdel-Naby, A. M. S. Ismail, A. M. Abdel-Fattah, A. F. Abdel-Fattah, Preparation and some properties of immobilized Penicillium funiculosum 258

U

dextranase. Process Biochem. 34 (1999) 391-398.

A

Properties. ACS Nano 8 (2014) 9733-9754.

N

[34] R. L. D. Whitby, Chemical Control of Graphene Architecture: Tailoring Shape and

[35] D. Wang, H. Duan, J. Lu, C. Lu, Fabrication of thermo-responsive polymer

M

functionalized reduced graphene oxide@Fe3O4@Au magnetic nanocomposites for

ED

enhanced catalytic applications. J. Mater. Chem. A 5 (2017) 5088-5097. [36] V. Georgakilas, J. N. Tiwari, K. C. Kemp, J. A. Perman, A. B. Bourlinos, K. S. Kim, R. Zboril, Noncovalent Functionalization of Graphene and Graphene Oxide for

PT

Energy Materials, Biosensing, Catalytic, and Biomedical Applications. Chem. Rev. 116 (2016) 5464-5519.

CC E

[37] J. Lee, H. Park, W. J. Kim, Nano "Chocolate Waffle" for near-IR Responsive Drug Releasing System. Small 11 (2015) 5315-5323. [38] J.-F. Huang, J. Zhong, G.-P. Chen, Z.-T. Lin, Y. Deng, Y.-L. Liu, P.-Y. Cao, B.Wang,

A

Wei, Y.; T. Wu, J. Yuan, G.-B. Jiang, A Hydrogel-Based Hybrid Theranostic Contact Lens for Fungal Keratitis. ACS Nano 10 (2016) 6464-6473. [39] Z. C. Xiong, H. Q. Qin, H. Wan, G. Huang, Z. Zhang, J. Dong, L. Y. Zhang, W. B. Zhang, H. F. Zou, Layer-by-layer assembly of multilayer polysaccharide coated magnetic nanoparticles for the selective enrichment of glycopeptides. Chem. Commun. 49 (2013) 9284-9286. 28

[40] G. Xu, X. Chen, J. Hu, P. Yang, D. Yang, L. Wei, Immobilization of trypsin on graphene oxide for microwave-assisted on-plate proteolysis combined with MALDIMS analysis. Analyst 137 (2012) 2757-2761. [41] C. Guo, X. Zhao, W. Zhang, H. Bai, W. Qin, H. Song, X. Qian, Preparation of polymer brushes grafted graphene oxide by atom transfer radical polymerization as a new support for trypsin immobilization and efficient proteome digestion. Anal. Bioanal.

IP T

Chem. 409 (2017) 4741-4749.

[42] Y. Weng, B. Jiang, K. Yang, Z. Sui, L. Zhang, Y. Zhang, Polyethyleneimine-

SC R

modified graphene oxide nanocomposites for effective protein functionalization. Nanoscale 7 (2015) 14284-14291.

[43] B. Jiang, K. Yang, Q. Zhao, Q. Wu, Z. Liang, L. Zhang, X. Peng, Y. Zhang,

U

Hydrophilic immobilized trypsin reactor with magnetic graphene oxide as support for

N

high efficient proteome digestion. J. Chromatogr. A 1254 (2012) 8-13.

A

[44] M. Royvaran, A. Taheri-Kafrani, A. L. Isfahani, S. Mohammadi, Functionalized superparamagnetic graphene oxide nanosheet in enzyme engineering: A highly

M

dispersive, stable and robust biocatalyst. Chem. Eng. J. 288 (2016) 414-422.

ED

[45] S. K. S. Patel, S. H. Choi, Y. C. Kang, J.-K. Lee, Eco-Friendly Composite of Fe3O4Reduced Graphene Oxide Particles for Efficient Enzyme Immobilization. ACS Appl. Mater. Interfaces 9 (2017) 2213-2222.

PT

[46] H. Wang, F. Jiao, F. Gao, X. Zhao, Y. Zhao, Y. Shen, Y. Zhang, X. Qian, Covalent organic framework-coated magnetic graphene as a novel support for trypsin

CC E

immobilization. Anal. Bioanal. Chem. 409 (2017) 2179-2187. [47] T. Zeng, X.-L, Zhang, Y. R. Ma, H. Y. Niu, Y. Q. Cai, A novel Fe3O4-graphene-Au multifunctional nanocomposite: green synthesis and catalytic application. J. Mater.

A

Chem. 35 (2012) 18658-18663. [48] D. Wang, M. Lu, S. Wang, Y. Jiao, W. Li, Q. Zhu, Z. Liu, Purification and characterization of a novel marine Arthrobacter oxydans KQ11 dextranase. Carbohyd. Polym. 106 (2014) 71-76. [49] M. Bashari, M. H. Abdelhai, S. Abbas, A. Eibaid, X. Xu, Z. Jin, Effect of ultrasound and high hydrostatic pressure (US/HHP) on the degradation of dextran 29

catalyzed by dextranase. Ultrason. Sonochem. 21 (2014) 76-83. [50] Q. Wu, B. Jiang, Y. Weng, J. Liu, S. Li, Y. Hu, K. Yang, Z. Liang, L. Zhang, Y. Zhang, 3-Carboxybenzoboroxole Functionalized Polyethylenimine Modified Magnetic Graphene Oxide Nanocomposites for Human Plasma Glycoproteins Enrichment under Physiological Conditions. Anal. Chem. 90 (2018) 2671-2677. [51] G. Baskar, N. A. Banu, G. Leuca, V. Gayathri, N. Jeyashree, Magnetic

IP T

immobilization and characterization of -amylase as nanobiocatalyst for hydrolysis of sweet potato starch. Biochem. Eng. J. 102 (2015) 18-23.

SC R

[52] F. A. Erhardt, H. J. Jordening, Immobilization of dextranase from Chaetomium erraticum. J. Biotechnol. 131 (2007) 440-447.

[53] M. Amirbandeh, A. T. Kafrani, A. Soozanipour, C. Gaillard. Triazine-

U

functionalized chitosan-encapsulated superparamagnetic nanoparticles as reusable and

N

robust nanocarrier for glucoamylase immobilization. Biochem. Eng. J. 127 (2017) 119-

A

127.

[54] S. Rouhani, A. Rostami, A. Salimi, O. Pourshiani, Graphene oxide/CuFe2O4

M

nanocomposite as a novel scaffold for the immobilization of laccase and its application

ED

as a recyclable nanobiocatalyst for the green synthesis of arylsulfonyl benzenediols. Biochem. Eng. J. 133 (2018) 1-11.

[55] Y. J. Jiang, J. Q. Zhai, L. Y. Zhou, Y. He, L. Ma, J. Gao. Enzyme@silica hybrid

PT

nanoflowers shielding in polydopamine layer for the improvement of enzyme stability.

A

CC E

Biochem. Eng. J. 132 (2018) 196-205.

30