Xylanase Immobilized on Novel Multifunctional Hyperbranched

Page 1 of 37

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

Langmuir

Graphical Abstract 253x159mm (150 x 150 DPI)

ACS Paragon Plus Environment

Langmuir

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

Page 2 of 37

Xylanase Immobilized on Novel Multifunctional Hyperbranched Polyglycerol-Grafted Magnetic Nanoparticles: An Efficient and Robust Biocatalyst Amir Landarani-Isfahani,†* Asghar Taheri-Kafrani,‡* Mina Amini,† Valiollah Mirkhani,† Majid Moghadam,† Asieh Soozanipour,‡ and Amir Razmjou‡ †

Catalysis Division, Department of Chemistry, University of Isfahan, Isfahan 81746-73441, Iran

‡

Department of Biotechnology, Faculty of Advanced Sciences and Technologies, University of Isfahan, Isfahan 81746-73441, Iran

Corresponding Authors A. Taheri-Kafrani

A. Landarani Isfahani

[email protected]

[email protected]

Tel: +98 31 37 93 43 46

Tel: +98 31 37 93 27 15

Fax: +98 31 37 93 23 42

Fax: +98 31 36 68 97 32


1

Page 3 of 37

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

Langmuir

ABSTRACT

Although several strategies are now available for immobilization of enzymes to magnetic nanoparticles for bio-applications, little progresses have been reported on the use of dendritic or hyperbranched polymers for the same purpose. Herein, we demonstrated synthesis of magnetic nanoparticles supported hyperbranched polyglycerol (MNP/HPG) and a derivative conjugated with citric acid (MNP/HPG-CA), as unique and convenient nano platforms for immobilization of enzymes. Then, an important industrial enzyme, xylanase, was immobilized on the nanocarriers to produce robust biocatalysts. A variety of analytical tools was used to study the morphological, structural and chemical properties of the biocatalysts. Additionally, the results of biocatalyst systems exhibited the substantial improvement of reactivity, reusability and stability of xylanase due to this strategy, which might confer them a wider range of applications.


2

Langmuir

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

Page 4 of 37

INTRODUCTION Nowadays, enzymes are widely used as biocatalysts. They are accepted to be more economical and environmental friendly, due to their high efficiency, low cost, excellent chemo-, regio-, and stereo-selective. Besides they operate in shorter synthetic routes compared with the chemical catalysts.1 Notwithstanding all these advantages, the practical industrial application of enzymes in various processes are often limited by the unsatisfactory operational and storage stability, high sensitivity to the environmental conditions, the extensive incubation time and the difficulties in recovery or reuse.2,3 To overcome the above mentioned limitations, immobilization of enzymes on various supports is considered to be an impressive and economical strategy to improve the stability and facilitate separation from reaction systems. Over the years, various natural and synthetic materials have been developed as efficient supports for enzyme immobilization, such as organic membrane, silicon matrix, and nanomaterials.4-7 Among them, nanomaterials have attracted more attention than other bulk materials, because they provide large surface area for immobilization of the enzyme. Hence, nano-size supports have been prospected as the promising carriers for enzyme stabilization.8-12 In the past few years, magnetic nanoparticles have attracted researchers due to their potential applications

in

catalysis,

electrochemistry,

sensors

and

biotechnology.13–18

Magnetic

nanoparticles modified by polymers are considered to be an interesting alternative for enzyme immobilization, due to their outstanding advantages and properties, including high loading capacity and favorable biocompatibility. Besides they are easily isolated from the reaction mixtures and can be recycled for reuse.19-21 Until now, a range of polymer brushes, such as poly(acrylic acid), chitosan, poly(ethylene glycol) and poly(glycidyl methacrylate), have been successfully employed to modify the surface of magnetic nanoparticles for fabrication of the


3

Page 5 of 37

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

Langmuir

immobilized enzyme with high activity and stability.22-24 However, rigorous reaction condition, long reaction times, low immobilization efficiency of enzyme, low solubility and leaching of enzymes are reported as the main limitations in the previously introduced approaches.25-28 Therefore, exploration and fabrication of novel polymer-modified magnetic nanocluster for convenient and efficient enzyme immobilization is in high demand. Recently, magnetic nanoparticles grafted with hyperbranched polyglycerol (MNP/HPG) have attracted more interest due to their interesting three dimensional structure and special chemical and physical properties.29,30 Moreover, HPG-modified magnetic nanomaterials could be used in biomedical applications, including drug delivery and magnetic resonance imaging (MRI), since they present biocompatibility properties, high dispersion and hydrophilicity.30-33 Surprisingly, as far as we know, no reports on the enzyme immobilization by the HPG-modified magnetic nanocarriers can be found. However, immobilization of enzymes on HPG-magnetic nanoparticles can promote the generation of new hyperhydrophilic microenvironment. This new hyperhydrophilic microenvironment can strongly improve the stability as well as the catalytic behavior of the immobilized enzyme.34-37 As one of the most significant enzymes for biotechnological approaches, xylanases (1,4-β-xylan xylanohydrolase; EC 3.2.1.8) are wildely used for a great variety of applications, such as food and beverages (bakery goods, coffee, starch, plant oil and juice manufacture), feedstock improvement (increasing animal feed digestibility) and the quality improvement of lignocellulosic residues.38-43 The immobilization of xylanase on various supports is considered to be an effective strategy to improve its stability and ensure its easy separation from the reaction systems. Thanks to many years of efforts from various research groups, different kinds of functionalized adsorbents have been developed to immobilize xylanase.44-46 Unfortunately, the


4

Langmuir

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

Page 6 of 37

absorbed xylanase is often easily leached out from the solid supports, resulting in xylanase loss and poor operational stability and reactivity.28,44-47 Therefore, solid support/nanoparticles functionalized with appropriate organic functional groups for immobilization of xylanase with high activity and stability still remains a big challenge. Motivated by the unique properties and many applications of nanomagnetic supported enzymes, herein, we explore HPG-modified magnetic nanoparticles (MNP/HPG) and a derivative conjugated with citric acid (MNP/HPG-CA) for high loading capacity, catalytic activity, stability and reusability of the immobilized xylanase, which can be extended to the immobilization of other enzymes.

EXPERIMENTAL SECTION General Remarks. The chemicals used in this work were obtained from Merck and Aldrich chemical companies. Xylanase (EC 3.2.1.8 from Thermomyces lanuginosus, ≥2500 units/g), beechwood xylan, and xylose were purchased from Sigma-Aldrich. The protein assay standard was bovine serum albumin (BSA), obtained from Bio-Rad (USA). DNS (3,5-dinitrosalicylic acid) was purchased from Fluka. Fourier transform infrared (FT-IR) spectra were recorded on a JASCO 6300 spectrophotometer. Thermogravimetric analysis (TGA) were carried out on a Mettler TG50 instrument, under air flow, at an uniform heating rate of 5 ◦C/min in the range of 30-600 ◦C. Field emission scanning electron microscopy (FE-SEM) was performed on a Hitachi S-4160 field emission-scanning electron microscope. The transmission electron microscopy (TEM) was carried out on a Philips CM30 transmission electron microscope, operating at 200 kV. X-ray powder diffraction (XRD) spectra were taken on a Bruker D8-advance x-ray diffractometer with Cu Kα radiation. Measurements of the magnetic properties of the samples


5

Page 7 of 37

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

Langmuir

have been carried out on alternating gradient force magnetometer from Meghnatis Daghigh Kavir Co. Synthesis of HPG-Grafted on Silica-Encapsulated Magnetic Nanoparticles. The Fe3O4 nanoparticles were prepared and coated with silica, according to the reported procedures.48 In a glove box, containing N2 atmosphere, a saturated solution of potassium methoxide in methanol (2 mL) was mixed with 0.100 g of silica coated magnetic nanoparticles (MNP). The mixture was sonicated in an ultrasonic bath for 30 min and stirred at room temperature for 1 h. Then it was refluxed at 80 ◦C for 2 h. At the end of the reaction, the magnetic nanoparticles were separated by a magnet, washed three times with dry methanol and dried in a vacuum oven at 60 ◦C. The glycidol (2 mL) was added slowly to these deprotonated nanoparticles at 100 ◦C and the mixture was stirred at 100 ◦C for 4 h. Then it was cooled and the contents were dissolved in methanol. Subsequently the product was separated by a magnet and washed with methanol under sonication. After repeated washing and separation steps, the resulting solid was dried overnight, under a vacuum, to obtain the HPG-grafted on silica-encapsulated magnetic nanoparticles (MNP/HPG). Synthesis of MNP/HPG-TDI. A sonicated mixture of MNP/HPG (100 mg) in dry toluene (3 ml) was added, dropwise, to a solution of 2,4-toluenediisocyanate, TDI (1 mL), in dry toluene (2 mL) under N2 atmosphere and the mixture was stirred at 75 ◦C for 24 h. After cooling to room temperature, the product was collected by a magnet and washed with anhydrous toluene several times and dried under vacuum. Synthesis of MNP/HPG-CA. A solution of citric acid, CA, (1.47 mL) in dry THF was mixed with MNP/HPG/TDI (100 mg) and the mixture was refluxed for 48 h. Then, it was cooled to


6

Langmuir

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

Page 8 of 37

room temperature and the product was separated by a magnet and washed with THF several times. Finally, the separated product was dried in a vacuum oven at 50 ◦C for 24 h. Immobilization of Xylanase onto MNP/HPG and MNP/HPG-CA. In a round bottom flask, five milligrams of MNP/HPG or MNP/HPG-CA were dispersed in 2 mL of phosphate buffer (20 mM, pH 6.5). Then, various volumes of the xylanase solution with a concentration of 4 mg/mL were added into the suspension and the mixture was shaken at room temperature for 8 h. The xylanase immobilized on MNP/HPG (MNP/HPG/Xy) or MNP/HPG-CA (MNP/HPG-CA/Xy) were separated from free xylanases by magnetic separation, and washed three times with phosphate buffer (20 mM, pH 6.5). The amount of the enzymes immobilized on MNP/HPG or MNP/HPG-CA nanocarriers were determined by measuring the initial and final concentrations of xylanase in the immobilization medium, using the Bradford protein assay.49 In this method, the amount of protein in the supernatant was determined colorimetrically (595 nm) with the Bio-Rad protein assay reagent concentrate using BSA as the standard protein. The amount of bound enzymes onto nanocarriers was calculated from Eq. (1): Immobilization efficiency % =

× 100

(1)

where Ci and Cs are the concentrations of xylanase initially used for reaction, and the unbound xylanase collected in each purification cycle, respectively. Xylanase Activity Assay. Activities of immobilized xylanase were assayed according to the method reported by Bailey, with minor modification.50 Using xylan as the substrate, the xylanase activity was evaluated by calculating the rate of the reducing sugars (xylose equivalent) production under the standard conditions and using 3,5-dinitrosalicylic acid reagent.51 Practically, a xylan solution (1.0%, w/v) was prepared by dissolving beechwood xylan in 20 mM phosphate buffer, pH 6.5. An appropriate amount of either immobilized or free xylanase were


7

Page 9 of 37

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

Langmuir

incubated (under shaking) at 60 ◦C for 10 min. The reaction was terminated by adding 3,5dinitrosalicylic acid reagent (DNS). After boiling for 5 min, the absorbance of reducing sugars released in the reaction was measured at 530 nm (Carry-500 double beam spectrophotometer). Control experiments without any enzyme were also performed. No xylose was generated when just MNP/HPG or MNP/HPG-CA were used as catalysts. The xylose equivalents were obtained by reference to the standard curve. All measurement experiments were carried out three times, and the experimental error was less than 3%. One unit (IU) of xylanase activity was defined as the amount of enzyme catalyzed the release of 1 µmol of reducing sugar as xylose equivalent per minute under the specified assay conditions. The activity recovery of the immobilized enzyme is calculated from the Eq. (2): % =

× 100%

(2)

where R is the activity recovery of the immobilized enzyme (%), A is the activity of the immobilized enzyme (U), and A0 is the activity of the free enzyme in solution before immobilization (U). Optimum Conditions of Immobilized Xylanase Activity. The activity of immobilized xylanase was assessed at different temperature and pH values by the enzyme assay, as described above. The optimum pH for the activity of both MNP/HPG/Xy and MNP/HPG-CA/Xy was determined by performing the assays at 60 ◦C with different pH values (3.5, 4.5, 5.5, 6.5, 7.5 and 8.5). The optimum temperature for soluble and immobilized enzyme was determined by assaying the enzyme activity at temperature ranges variation from 40 to 90 ◦C, with 5 ◦C intervals, in 20 mM phosphate buffer, pH 6.5. Reusability of Immobilized Xylanase. The reusability was determined by incubating the immobilized xylanase at the same conditions. After one reaction was over, the same immobilized


8

Langmuir

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

Page 10 of 37

enzyme was removed from the reaction medium, with a conventional permanent magnet, thoroughly rinsed with phosphate buffer (20 mM, pH 6.5) and then, added to a fresh substrate solution to start a new enzymatic reaction cycle. The specific activity of the first run was set to 100%. Storage Stability. For storage stability studies, the immobilized xylanase on MNP/HPG and MNP/HPG-CA was kept at 4 ◦C in 20 mM phosphate buffer, pH 6.5. The activity of immobilized enzyme was determined using the assay procedures described above. The residual activity of immobilized xylanase was measured every 15 days for a period of 100 days.

RESULTS AND DISCUSSION Synthesis and Characterization. A schematic illustration of the step-by-step approach used for the preparation of the magnetic nanoparticles-immobilized xylanase, is shown in Scheme 1. First, the super paramagnetic nanoparticles (Fe3O4 NPs) were synthesized by a reported coprecipitation method. Then, the Fe3O4 NPs were coated with silica through hydrolysis of tetraethylorthosilicate (TEOS), according to the Stöber method, to obtain silica coated magnetic nanoparticles (MNP). The silica coating reaction is a simple, convenient and valuable method due to its ability to avoid the aggregation and chemical degradation of magnetic nanoparticles in harsh conditions. Besides, the surface Si-OH groups can be used to initiate the ring-opening polymerization of glycidol, affording HPG-functionalized magnetic nanoparticles (MNP/HPG) (Scheme 1). The reactions were screened by FT-IR spectra, TGA and elemental analysis. Figure S1 shows the FT-IR spectrum for the Fe3O4 NP, the silica coated magnetic NPs (MNP) and the MNP/HPG. In the FT-IR spectra (Figure S1a), the band at about 590 cm-1 was assigned to the vibration of the


9

Page 11 of 37

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

Langmuir

Fe-O bonds. Furthermore, there was an obvious band at 3435 cm-1 attributed to the –OH groups, demonstrating the existence of Fe3O4 components. Additionally, the bands at 1089 and 806 cm-1 were attributed to Si-O vibration, indicating the existence of SiO2 shell (Figure S1b). As shown in Figure S1c, the appearance of new bands at 2895 and 2917 cm-1, related to the absorption of C-H bond, confirms the synthesis of MNP/HPG nanocarrier. The thermal stability and weight loss of the MNP/HPG were also evaluated by TGA. According to the TGA curves (Figure S2a), about 4 wt% weight loss was observed for MNP below 600 °C. The TGA curve of MNP/HPG (Figure S2b) represents two weight-loss steps. The first step (between 30-200 °C) is assigned to the removal of physically adsorbed solvents, while the second one (200-600 °C) is related to the loss of polymer chain. As shown in Figure S2b, after the polymerization, the weight loss was 25.2 wt% in the temperature range of 200-600 °C, confirming the successful modification of MNPs by HPG polymers (Figure S2b). The carbon content of MNP/HPG, determined by CHN analysis, was found to be 10.7 wt%. Based on these results, it can be estimated that the glycerol loading of MNP is about 2.9 mmol/g. To show the versatility of the reactive platform of MNP/HPG, the MNP/HPG-TDI particles were treated, sequentially, with 2,4-toluene diisocyanate (TDI) and citric acid, to form MNP/HPG-CA (Scheme S1). These successful post-functionalizations were also confirmed by FT-IR spectra, TGA and elemental analysis. The TDI bearing two isocyanate (NCO) groups, one of the NCO groups reacted and the other remained intact (MNP/HPG-TDI). The presence of the absorption band at 2276 cm-1 in FT-IR spectrum of MNP/HPG-TDI, is a good indication for the presence of NCO groups. As shown in Figure S3a, the bands that appeared at 1400-1500 cm-1 and also at 1585-1600 cm-1 corresponded to C-C stretching vibrations of the aromatic rings, and the bands at 650-900 cm-1 associated with


10

Langmuir

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

Page 12 of 37

the aromatic out-of-plane C-H bending. In addition, the band at 3321 cm-1 corresponded to the vibration of the NH bond and the bands at 570 cm-1 were assigned to the vibration of the Fe-O bonds. Other peaks at 1080, 812 and 470 cm-1 corresponded to the asymmetric stretching, symmetric and bending vibration of Si-O-Si bonds, respectively, and the bands at 2870 and 2922 cm-1 were associated to –CH2 stretching. After reaction of the NCO group with citric acid, the absorption bands corresponding to the acidic groups, including the carbonyl group at 1716 cm-1 and the OH absorption band at 3030-3720 cm-1, appeared (Figure S3b). These observations confirmed the successful synthesis of MNP/HPG-CA. The TGA technique was used to confirm the synthesis of citric acid modified nanocarrier. As shown in Figure S2c, the weight loss for MNP/HPG-CA between 200-600 ᵒC was increased to 41.4 wt%, suggesting the successful post-functionalization. Based on the results of elemental analysis, the nitrogen content of the MNP/HPG/TDI was found to be 2.74 wt%, demonstrating the existence of TDI components. The XRD pattern of the MNP/HPG-CA is shown in Figure 1. A broad peak, around 2θ of 20-30°, can be observed. It corresponds to amorphous phase of SiO2.

Bisides, the characteristic

diffraction peaks, at 2θ = 30.09, 35.44, 43.07, 54.43, 57.16 and 63.55, are in agreement with face centered cubic (fcc) Fe3O4. After preparation and characterization of MNP/HPG-CA, xylanase was immobilized onto this nanostructure platform (Scheme S2). The morphology of the surface of nanostructure platform and immobilized xylanase were studied by FE-SEM (Figure 2). This technique was used to visualize directly the size and surface morphology of MNP/HPG-CA and MNP/HPG-CA/Xy. As can be seen in this figure, particles are spherical and have diameters in the range of nanometers. The small dimensions of


11

Page 13 of 37

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

Langmuir

nanoparticles provided larger specific surface to volume ratio and subsequently higher efficient enzyme immobilization. Further characterizations of the magnetic nanocrriers were performed by TEM. As shown in Figure 3, the TEM images of MNP/HPG-CA/Xy clearly display that Fe3O4 nanoparticles have been successfully encapsulated into the polymer shell. The dark core relates to MNPs, while after functionalization of magnetic nanoparticles, the light shell corresponds to polymer shell. Magnetic measurements of MNP, MNP/HPG, MNP/HPG-CA, MNP/HPG/Xy and MNP/HPGCA/Xy were investigated by a SQUID magnetometer, at 300 K, in an applied magnetic field, ranging from -8000 to 8000 Oe (Figure S4). The results demonstrated that the saturation magnetization (Ms) values of MNP, MNP/HPG, MNP/HPG-CA, MNP/HPG/Xy and MNP/HPGCA/Xy were 46.3, 25.8, 19.6, 13.2, and 9.6 emu/g, respectively. The gradual decline in magnetic response implied an increase of thickness of the shell layer on the MNPs surface during the functionalization procedure. Accordingly, the amount of Ms for MNP/HPG and MNP/HPG-CA were found to be considerably lower than MNP, due to the functionalization of silicaencapsulated Fe3O4. These results demonstrated that by increasing the polymer shell, the saturation magnetization of nanocarriers decreased. The immobilization of xylanase on the MNP/HPG and MNP/HPG-CA surfaces caused in the reduction of their magnetization properties. In addition, the magnetic separation capability of prepared MNP/HPG/Xy and MNP/HPG-CA/Xy was tested in an aqueous solution, by placing an external magnet close to the glass bottle. The suspended particles could be easily collected by the magnet within 1 min. The results confirmed that, even with this reduction in the saturation magnetization, the solid can still be easily separated from the solution with a magnet and immediately redispersed, after removing the magnetic field.


12

Langmuir

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

Page 14 of 37

Reaction Conditions Optimization and Activity Assay. Compared to free enzyme, the immobilization of enzymes usually leads to a higher stability but the trade-off is a reduction in activity due to the immobilization procedure and mass transfer limitations.52-54 Before conducting experiments and also to find the optimal reaction conditions, the activity of free and immobilized enzyme were measured. Xylanase was immobilized at pH 6.5, on MNP/HPG and MNP/HPGCA, in order to have MNP/HPG/Xy and MNP/HPG-CA/Xy biocatalysts, respectively. The efficiency of immobilization was assayed based on the amounts of xylanase which was immobilized on the nanocarriers of uniform mass. The Bradford method was used to determine the amount of xylanase in the solution after immobilization.49 Figure 4a shows the effect of addition of enzyme on the activity recovery of the immobilized enzyme. The activity recovery of the immobilized xylanase on the MNP/HPG-CA nanocarrier reached 89.7% and was higher than the activity recovery (78.2%) of that on the MNP/HPG nanocarrier. The amounts of xylanase loaded on nanocarriers were about 236 and 279 mg of enzyme per MNP/HPG and MNP/HPGCA nanocarriers gram, respectively (Figure 4a). The effect of reaction time on the activity of immobilized xylanase is presented in Figure 4b. As shown in this figure, by increasing the reaction time from 1 to 8 h, the amount of immobilized xylanases on nanocarriers and their relative activity increased, remaining almost constant after about 8 hours. Therefore, the enhancement in the activity recovery of immobilized xylanase was observed with prolonged reaction time. The highest activity was obtained when the immobilization procedure allowed to proceed for 8 h. Consequently, the optimal conditions for the immobilization of xylanase on MNP/HPG and MNP/HPG-CA nanocarriers were 8 h time, room temperature, 20 mM phosphate buffer, pH 6.5. The activity recovery of MNP/HPG-CA/Xy was significantly higher than


13

Page 15 of 37

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

Langmuir

MNP/HPG/Xy, when a further reaction time was allowed. This phenomenon occurred due to the difference on the structures of the supports. Using xylan as substrate the catalytic activity of xylanase before and after immobilization were evaluated under optimum conditions (a temperature of 65 °C, pH 6.5). Figure 5a shows the effect of the pH on the activity of the immobilized enzyme. As shown in this figure, the activity recovery of the immobilized enzyme on the MNP/HPG-CA reached about 88% and was clearly higher than the activity recovery (about 74%) of that on the MNP/HPG at 65 °C and pH 6.5, which demonstrated that the immobilized xylanase on the MNP/HPG-CA showed a great degree of adaptability in a wider range of pH, when compared with that of HPG grafted MNPs. This alteration in activity through pH changes probably comes from the characteristic of the nanocarriers. The impact of reaction temperature on the activity recovery of immobilized enzymes is presented in Figure 5b. In comparison with the MNP/HPG, the immobilized xylanase showed the higher activity recovery on the MNP/HPG-CA nanocarriers, within 55–75 °C, which indicated that the immobilized xylanase on citric acid modified nanocarriers has a wider temperature endurance ranges. Also, it can be seen that the activity recovery of the MNP/HPGCA/Xy could reach about 89%, and was clearly higher than the activity recovery (about 76%) of xylanase immobilized on the MNP/HPG at 65 °C, 20 mM phosphate buffer, pH 6.5. In conclusion, the optimum temperature and pH range for the activity of MNP/HPG/Xy and MNP/HPG-CA/Xy became wider, at the same time, compared to the free xylanase. In other words, a higher environmental tolerance (temperature and pH) was observed for xylanase after immobilization on the MNP/HPG and MNP/HPG-CA by non-covalent attachment. Reusability of Immobilized Xylanase. One of the main concerns that can limit the commercial implementation of any immobilized enzyme is the reusability of immobilized enzymes.


14

Langmuir

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

Page 16 of 37

Nanocarriers can provide a new catalytic nanoenvironment and also facilitate reusability for easy separation. The incorporation of magnetic technology with enzyme immobilization on the nanocarriers can improve the recoverability and reusability of the biocatalysts.55 In order to examine the reusability, the xylanase-immobilized on MNP/HPG and MNP/HPG-CA were washed after repeated runs and were reintroduced into a fresh solution, using the optimum conditions (65 ᵒC and pH 6.5). The first run was a control and its specific activity was set to 100%. To evaluate the catalytic activities of the artificial nanosystems, the yield of the final target product (xylose) was considered. The immobilized enzyme was used for 10 reaction cycles in the reusability experiments. As shown in Figure 6, the xylanase immobilized on citric acid modified HPG nanocarriers could retain around 66% of its initial activity after 10 cycles of reuse; whereas under the same conditions, the MNP/HPG/Xy nanocarriers could retain only 54% of the original activity. The main cause of activity loss is related to the leaching of enzyme which is due to the lack of binding strength between the immobilized enzyme and the matrix caused by repeated use. Furthermore, the recurrent encountering of substrate with the active site of immobilized enzyme causes its distortion, and leads to loss of activity. The obvious reusability differences between MNP/HPG and MNP/HPG-CA might be attributed to different interactions connecting the enzymes and the matrix, in which the citric acid modified HPG nanocarriers with COOH groups have more strong interaction with xylanase, compared with HPG matrix with OH groups. Kinetic Results. The success of immobilization process can be monitored by investigating the changes in the kinetic parameters of the enzyme (Km and vmax) due to immobilization. The activity of enzyme as a function of xylan concentration (0.5-20 mg/mL), as substrate, was measured for free and immobilized xylanase, using standard assay procedures. The results was


15

Page 17 of 37

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

Langmuir

analyzed using Lineweaver-Burk plot to obtain the Michaelis–Menten constant (Km) and the maximum velocity (vmax). The kinetic parameters of the free and immobilized xylanases were calculated and compared to each other (Table 1). The values of Km for MNP/HPG/Xy, MNP/HPG-CA/Xy and free xylanase were found to be 5.27 mg/mL, 4.83 mg/mL and 4.16 mg/mL, respectively. The MNP/HPG/Xy, MNP/HPG-CA/Xy biocatalysts demonstrated Km values slightly higher than free enzyme. The vmax values were found to be 1.18, 1.33 and 1.79 U/mL for MNP/HPG/Xy, MNP/HPG-CA/Xy and free xylanase, respectively. The increase in the Km values of the immobilized enzyme could be due to steric effects which are a result of limitation of the accessibility of substrate to the active site of the enzyme after immobilization. Besides, the conformational changes in tertiary structure of enzyme due to immobilization can be a further explanation of decrease in the catalytic efficiency and increase in Km values.56 Moderate increases in Km of xylanase subsequent to immobilization is previously reported.28,46,57-58 As a result, the maximum rate of the reaction which was catalyzed by immobilized xylanase was lower than that of the free xylanase. The values kcat for MNP/HPG/Xy, MNP/HPG-CA/Xy and free xylanases were calculated (Table 1). When compared with the free xylanase, the kcat values of the immobilized xylanase on MNP/HPG and MNP/HPG-CA were decreased by 17% and 9%, respectively. The reduced number of active sites after xylanase immobilization on MNP/HPG and MNP/HPG-CA, should be responsible for the decrease in kcat.59 This downswing was more obvious in MNP/HPG/Xy which could be due to weaker interaction of the enzyme with matrix, in comparison with xylanase immobilized on citric acid modified HPG nanocarriers. Storage Stability. One of the key factors which often affects the final assessment and selection of any industrially important enzyme over other available enzymes in the similar domain is storage stability. Having an enzyme with excellent storage stability without losing substantial


16

Langmuir

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

Page 18 of 37

biocatalytical efficiency is desirable. In order to investigate the storage stability of free and immobilized enzymes, they were kept at 4 °C in phosphate buffer (20 mM, pH 6.5) for 100 days. As can be seen in Figure 7, the activity of soluble xylanase reduced much faster than those of the immobilized enzymes under the same storage conditions. The xylanase immobilized on MNP/HPG and MNP/HPG-CA retained about 67% and 74% of its initial activity after 100 days storage at the same conditions, respectively. In contrast, the free xylanase only retained about 46% of its original activity over the same period of time and condition. The reason may be connected with improved resistance of the immobilized xylanase to conformational changes in solution, due to the neutralization of charged residues by the interaction with solid substrate or less significant exposure after immobilization. Accordingly, the possible distortion effects, which could be imposed by aqueous medium on the active site of xylanase, were decreased.60 The difference between the storage stability of the xylanase-immobilized on MNP/HPG and MNP/HPG-CA could be attributed to the special interactions involving the enzyme and matrix.

CONCLUSIONS In conclusion, we confirmed robust and convenient biocatalyst based on novel multifunctional hyperbranched polyglycerol-grafted magnetic nanoparticles for immobizliation of enzyme. Combining the advantages of easy separation of magnetic nanoparticles and special physical properties of heperbrached polyglycerol, herein, MNP/HPG and its derivative conjugated with citric acid (MNP/HPG-CA) were used for xylanase immobilization as unique biocatalysts. The Bradford protein assay results indicated that the xylanase loading amount on the MNP/HPG and MNP/HPG-CA were about 236 and 279 mg of protein per nanocarrier gram, respectively. Also the immobilized xylanases exhibited excellent catalytic activities at pH 6.5 and 65 °C and found


17

Page 19 of 37

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

Langmuir

to keep more than 85% of the activity of free xylanase. Howevere, compared to MNP/HPG/Xy, the MNP/HPG-CA/Xy illustrated more efficient biocatalytic properties. Notably, MNP/HPGCA/Xy showed a remarkable stability such that even after 10 reaction cycles its activity retains about 66% of the initial activity. The results of this study demonstrated that the enzymes immobilized on functionalized HPG grafted magnetic nanoparticles have an excellent thermal and storage stability, which suggests that this immobilization approach could create a new biotechnological horizon for this expensive biocatalyst as it becomes economically viable. We believe the use of functionalized MNP/HPG is a promising support which can be extended to other enzyme systems, and is the subject of our future work. The fascinating properties of MNP/HPG nanocarrier, whose structure can be orientated and its surface architecture engineered, provides great opportunities for biotechnological applications, particularly in the field of enzyme immobilization and medicine.

ACKNOWLEDGMENT The authors are grateful to the Center of Excellence of Research Council of the University of Isfahan for financial supports of this work.

ABBREVIATIONS HPG, hyperbranch polyglycerol; MNP, silica encapsulated magnetic nanoparticles; MNP/HPG, HPG grafted MNP; MNP/HPG-TDI, 2,4-toluenediisocyanate modified MNP/HPG; MNP/HPGCA, Citric acid modified MNP/HPG-TDI; MNP/HPG/Xy, xylanase immobilized on MNP/HPG; MNP/HPG-CA/Xy, xylanase immobilized on MNP/HPG-CA


18

Langmuir

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

Page 20 of 37

REFERENCES (1)

Held, M.; Schmid, A.; Van Beilen, J. B.; Witholt, B. Biocatalysis. Biological systems for the production of chemicals. Pure Appl. Chem. 2000, 72 (7), 1337−1343.

(2)

Stepankova, V.; Bidmanova, S.; Koudelakova, T.; Prokop, Z.; Chaloupkova, R.; Damborsky, J. Strategies for stabilization of enzymes in organic solvents. ACS Catal. 2013, 3 (12), 2823-2836.

(3)

Cowan, D. A.; Fernandez-Lafuente, R. Enhancing the functional properties of thermophilic enzymes by chemical modification and immobilization. Enzyme. Microb. Techol. 2011, 49 (4), 326-346.

(4)

Tran, D. N.; Balkus Jr, K. J. Perspective of recent progress in immobilization of enzymes. Acs Catal. 2011, 1 (8), 956-968.

(5)

Barbosa, O.; Torres, R.; Ortiz, C.; Berenguer-Murcia, A.; Rodrigues, R. C.; FernandezLafuente, R. Heterofunctional supports in enzyme immobilization: from traditional immobilization protocols to opportunities in tuning enzyme properties. Biomacromolecules 2013, 14 (8), 2433-2462.

(6)

Lu, J.; Toy, P. H. (2009). Organic polymer supports for synthesis and for reagent and catalyst immobilization. Chem. Rev. 2009, 109 (2), 815-838.

(7)

Lykourinou, V.; Chen, Y.; Wang, X. S.; Meng, L.; Hoang, T.; Ming, L. J.; Ma, S. Immobilization of MP-11 into a mesoporous metal–organic framework, MP-11@ mesomof: a new platform for enzymatic catalysis. J. Am. Chem. Soc. 2011, 133 (27), 10382-10385.

(8)

Kim, J.; Grate, J. W.; Wang, P. Nanostructures for enzyme stabilization. Chem. Eng. Sci. 2006, 61 (3), 1017-1026.


19

Page 21 of 37

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

Langmuir

(9)

Hwang, E. T.; Gu, M. B. Enzyme stabilization by nano/microsized hybrid materials. Eng. Life Sci. 2012, 13 (1), 49-61.

(10) Jesionowski, T.; Zdarta, J.; Krajewska, B. Enzyme immobilization by adsorption: a review. Adsorption 2014, 20 (5-6), 801-821. (11) Campbell, A. S.; Dong, C.; Meng, F.; Hardinger, J.; Perhinschi, G.; Wu, N.; Dinu, C. Z. Enzyme Catalytic Efficiency: A function of bio–nano interface reactions. ACS Appl. Mater. Interfaces 2014, 6 (8), 5393-5403. (12) Kudina, O.; Zakharchenko, A.; Trotsenko, O.; Tokarev, A.; Ionov, L.; Stoychev, G.; Minko, S. Titelbild: highly efficient phase boundary biocatalysis with enzymogel nanoparticles. Angew. Chem. 2014, 126 (2), 493–497. (13) Song, C. E.; Lee, S. G. Supported chiral catalysts on inorganic materials. Chem. Rev. 2002, 102 (10), 3495-3524. (14) Fraile, J. M.; García, J. I.; Mayoral, J. A. Noncovalent immobilization of enantioselective catalysts. Chem. Rev. 2008, 109 (2), 360-417. (15) Gao, J.; Gu, H.; Xu, B. Multifunctional magnetic nanoparticles: design, synthesis, and biomedical applications. Acc. Chem. Res. 2009, 42 (8), 1097–1107. (16) Baig, R. N.; Varma, R. S. Magnetically retrievable catalysts for organic synthesis. Chem. Commun., 2013, 49 (8), 752-770. (17) Saeedi, M. S.; Tangestaninejad, S.; Moghadam, M.; Mirkhani, V.; MohammadpoorBaltork, I.; Khosropour, A. R. Manganese porphyrin immobilized on magnetite nanoparticles as a recoverable nanocatalyst for epoxidation of olefins. Mater. Chem. Phys. 2014, 146 (1), 113-120.


20

Langmuir

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

Page 22 of 37

(18) Yang, H. H.; Zhang, S. Q.; Chen, X. L.; Zhuang, Z. X.; Xu, J. G.; Wang, X. R. Magnetitecontaining spherical silica nanoparticles for biocatalysis and bioseparations. Anal. Chem., 2004, 76 (5), 1316–1321 (19) Laurent, S.; Forge, D.; Port, M.; Roch, A.; Robic, C.; Vander Elst, L.; Muller, R. N. Magnetic iron oxide nanoparticles: synthesis, stabilization, vectorization, physicochemical characterizations, and biological applications. Chem. Rev. 2009, 110 (4), 2574-2574. (20) Zhu, Y. T.; Ren, X. Y.; Liu, Y. M.; Wei, Y.; Qing, L. S.; Liao, X. Covalent immobilization of porcine pancreatic lipase on carboxyl-activated magnetic nanoparticles: Characterization and application for enzymatic inhibition assays. Mater. Sci. Eng: C, 2014, 38, 278-285. (21) Cui, R.; Bai, C.; Jiang, Y.; Hu, M.; Li, S.; Zhai, Q. Well-defined bioarchitecture for immobilization of chloroperoxidase on magnetic nanoparticles and its application in dye decolorization. Chem. Eng. J. 2015, 259, 640-646. (22) Qin, W. J.; Song, Z. F.; Fan, C.; Zhang, W. J.; Cai, Y.; Zhang, Y. J.; Qian, X. H. Trypsin immobilization on hairy polymer chains hybrid magnetic nanoparticles for ultra fast, highly efficient proteome digestion, facile 18O-labeling and absolute protein quantification. Anal. Chem. 2012, 84 (7), 3138−3144. (23) Huang, S. H.; Juang, R. S. Biochemical and Biomedical Applications of Multifunctional Magnetic Nanoparticles: A Review. J. Nanopart. Res. 2011, 13 (10), 4411−4430. (24) Konwarh, R.; Karak, N.; Rai, S. K.; Mukherjee, A. K. Polymer-assisted iron oxide magnetic nanoparticle immobilized keratinase. Nanotechnology 2009, 20 (22), 225107. (25) Wang, C. F.; Zhou, G. W.; Li, Y. J.; Lu, N.; Song, H. B.; Zhang, L. Biocatalytic esterification of caprylic acid with caprylic alcohol by immobilized lipase on aminofunctionalized mesoporous silica. Colloids Surf. A, 2012, 406, 75-83.


21

Page 23 of 37

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

Langmuir

(26) Ponvel, K. M.; Lee, D.-G.; Woo, E.-J.; Ahn, I.-S.; Lee, C.-H. Immobilization of lipase on surface modified magnetic nanoparticles using alkyl benzenesulfonate. Korean J. Chem. Eng. 2009, 26 (1), 127-130. (27) Edward, V. A.; Pillay, V. L.; Swart, P.; Singh, S. Immobilization of xylanase from Thermomyces lanuginosus SSBP using Eudragit S-100: research in action. S. Afr. J. Sci. 2002, 98 (11 & 12), 553-554. (28) Gawande, P. V.; Kamat, M. Y. Preparation, characterization and application of Aspergillus sp. xylanase immobilized on Eudragit S-100. J. Biotechnol. 1998, 66 (2), 165-175. (29) Zhou, L.; Gao, C.; Xu, W. Robust Fe3O4/SiO2-Pt/Au/Pd magnetic nanocatalysts with multifunctional hyperbranched polyglycerol amplifiers. Langmuir 2010, 26 (13), 1121711225. (30) Zhao, L.; Chano, T.; Morikawa, S.; Saito, Y.; Shiino, A.; Shimizu, S.; Komatsu, N. Hyperbranched

polyglycerol‐grafted

superparamagnetic

iron

oxide

nanoparticles:

Synthesis, characterization, functionalization, size separation, magnetic properties, and biological applications. Adv. Funct. Mater. 2012, 22 (24), 5107-5117. (31) Zhou, L.; Gao, C.; Xu, W. Magnetic dendritic materials for highly efficient adsorption of dyes and drugs. ACS App. Mate. Interfaces 2010, 2 (5), 1483-1491. (32) Li, M.; Neoh, K. G.; Wang, R., Zong, B. Y.; Tan, J. Y.; Kang, E. T. Methotrexateconjugated and hyperbranched polyglycerol-grafted Fe3O4 magnetic nanoparticles for targeted anticancer effects. Eur. J. Pharm.Sci. 2013, 48 (1), 111-120. (33) Lv, Y.; Ding, G.; Zhai, J.; Guo, Y.; Nie, G.; Xu, L. A superparamagnetic Fe3O4-loaded polymeric nanocarrier for targeted delivery of evodiamine with enhanced antitumor efficacy. Colloids Surf. B. 2013, 110, 411-418.


22

Langmuir

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

Page 24 of 37

(34) Bickerstaff, G. F. Immobilization of Enzymes and Cells. Springer 2006. (35) Wilson, L.; Illanes, A.; Abián, O.; Pessela, B. C. C.; Fernández-Lafuente, R.; Guisán, J. M. Co-aggregation of penicillin g acylase and polyionic polymers: a simple methodology to prepare enzyme biocatalysts stable in organic media. Biomacromolecules 2004, 5, 852– 857. (36) Abian O.; Wilson, L.; Mateo, C.; Fernández-Lorente,G.; Palomo, J. M.; FernándezLafuente, R.; Guisán, J. M.; Re, D.; Tam, A.; Daminatti, M. Preparation of artificial hyperhydrophilic micro-environments (polymeric salts) surrounding immobilized enzyme molecules. New enzyme derivatives to be used in any reaction medium. J. Mol. Cat. B Enzymatic 2002, 19–20C, 295–303. (37) Fernández-Lafuente, R.; Rosell, C. M.; Guisán, J. M.; Caanan-Haden, L.; Rodes, L. Facile synthesis of artificial enzyme nano-environments via solid-phase chemistry of immobilized derivatives dramatic stabilization of penicillin acylase versus organic solvents. Enzyme Microb. Technol. 1999, 24, 96–103. (38) Manimaran, A.; Kumar, K. S.; Permaul, K.; Singh, S. Hyper production of cellulase-free xylanase by Thermomyces lanuginosus SSBP on bagasse pulp and its application in biobleaching. Appl. Microbiol. Biotechnol. 2009, 81 (5), 887-893. (39) Jiang, Z. Q.; Yang, S. Q.; Tan, S. S.; Li, L. T.; Li, X. T. Characterization of a xylanase from the newly isolated thermophilic Thermomyces lanuginosus CAU44 and its application in bread making. Lett. Appl. Microbiol. 2005, 41 (1), 69-76. (40) Butt, M. S.; Tahir-Nadeem, M.; Ahmad, Z.; Sultan, M. T. Xylanases and their applications in baking industry. Food Technol. Biotech. 2008, 46 (1), 22-31.


23

Page 25 of 37

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

Langmuir

(41) Diebold, G.; Mosenthin, R.; Piepho, H. P.; Sauer, W. C. Effect of supplementation of xylanase and phospholipase to a wheat-based diet for weanling pigs on nutrient digestibility and concentrations of microbial metabolites in ileal digesta and feces. J. Anim. Sci. 2004, 82 (9), 2647-2656. (42) Bajaj, B. K.; Manhas, K. Production and characterization of xylanase from Bacillus licheniformis P11 (C) with potential for fruit juice and bakery industry. Biocatal. Agric. Biotechnol. 2012, 1 (4), 330-337. (43) Biely, P. Microbial xylanolytic systems. Trends Biotechnol. 1985, 3 (11), 286-290. (44)

Sheldon, R. A.; van Pelt, S. Enzyme immobilization in biocatalysis: why, what and how. Chem. Soc. Rev. 2013, 42 (15), 6223-6235.

(45) Soozanipour, A.; Taheri-Kafrani, A.; Landarani Isfahani, A. Covalent attachment of xylanase on functionalized magnetic nanoparticles and determination of its activity and stability. Chem. Eng. J. 2015, 270, 235-243. (46) Yan, X.; Wang, X.; Zhao, P.; Xu, P.; Ding, Y. Xylanase immobilized nanoporous gold as a highly active and stable biocatalyst. Micropor. Mesopor. Mat. 2012, 161, 1-6. (47) Pal, A.; Khanum, F. Covalent immobilization of xylanase on glutaraldehyde activated alginate beads using response surface methodology: Characterization of immobilized enzyme. Process Biochem. 2011, 46 (6), 1315-1322. (48) Schätz, A.; Hager, M.; Reiser, O. Cu (II) Azabis (oxazoline) complexes immobilized on superparamagnetic magnetite@ silica nanoparticles: A highly selective and recyclable catalyst for the kinetic resolution of 1, 2 Diols. Adv. Func. Mat. 2009, 19 (13), 2109-2115.


24

Langmuir

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

Page 26 of 37

(49) Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976, 72 (1), 248254. (50) Bailey, M. J.; Biely, P.; Poutanen, K. Interlaboratory testing of methods for assay of xylanase activity. J. Biotech. 1992, 23 (3), 257-270. (51) Miller, G. L. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 1959, 31 (3), 426-428. (52) Kim, J.; Grate, J. W.; Wang, P. Nanobiocatalysis and its potential applications. Trends Biotech. 2008, 26 (11), 639-646. (53) Luckarift, H. R.; Spain, J. C.; Naik, R. R.; Stone, M. O. Enzyme immobilization in a biomimetic silica support. Nature Biotech. 2004, 22 (2), 211-213. (54) Mateo, C.; Grazu, V.; Palomo, J. M.; Lopez-Gallego, F.; Fernandez-Lafuente, R.; Guisan, J. M. Immobilization of enzymes on heterofunctional epoxy supports. Nature Protocols 2007, 2 (5), 1022-1033. (55) Fuertes, A. B.; Tartaj, P. Monodisperse carbon–polymer mesoporous spheres with magnetic functionality and adjustable pore size distribution. Small 2007, 3 (2), 275-279. (56) Goldstein, L. Methods in Enzymology, vol. 44, Academic Press, New York, 1976, pp. 397. (57) Liu, M. Q.; Dai, X. J.; Guan, R. F.; Xu, X. Immobilization of Aspergillus niger xylanase A on Fe3O4 -coated chitosan magnetic nanoparticles for xylooligosaccharide preparation. Catal. Commun. 2014, 55, 6-10. (58) Kapoor, M.; Kuhad, R. C. Immobilization of xylanase from Bacillus pumilus strain MK001 and its application in production of xylo-oligosaccharides. Appl. Biochem. Biotech. 2007, 142 (2), 125-138.


25

Page 27 of 37

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

Langmuir

(59) Qiu, H.; Xu, C.; Huang, X.; Ding, Y.; Qu, Y.; Gao, P. Immobilization of laccase on nanoporous gold: comparative studies on the immobilization strategies and the particle size effects. J. Phys. Chem. C 2009, 113 (6), 2521-2525. (60) Mateo, C.; Palomo, J. M.; Fernandez-Lorente, G.; Guisan, J. M.; Fernandez-Lafuente, R. Improvement of enzyme activity, stability and selectivity via immobilization techniques. Enzy. Microb. Tech. 2007, 40 (6), 1451-1463.


26

Langmuir

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

Page 28 of 37

Table 1. Kinetic parameters of free and immobilized xylanase. xylanase immobilized on xylanase immobilized on free xylanase MNP/HPG

MNP/HPG-CA

Km (mg/mL)

4.16

5.27

4.83

vmax (U/mL)

1.79

1.48

1.63

kcat (min-1)

89.6

74.1

81.4


27

Page 29 of 37

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

Langmuir

Figure Legends: Scheme 1. Preparation route for MNP/HPG-CA/Xy.

Figure 1. XRD pattern of MNP/HPG-CA.

Figure 2. FE-SEM images of (a) MNP/HPG-CA and (b) MNP/HPG-CA/Xy.

Figure 3. TEM images of MNP/HPG-CA/Xy.

Figure 4. (a) Effect of the amount of enzyme, and (b) Effect of reaction time, on the activity recovery of immobilized xylanase on MNP/HPG and MNP/HPG-CA nanocarriers in 20 mM phosphate buffer, pH 6.5 at room temperature.

Figure 5. Effect of (a) pH and (b) temperature, on the activity recovery of immobilized xylanase on MNP/HPG and MNP/HPG-CA nanocarriers.

Figure 6. The reusability comparison of MNP/HPG/Xy and MNP/HPG-CA/Xy.

Figure 7. The storage stability of free and immobilized xylanases at 4 °C.


28

Langmuir

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

Page 30 of 37

Scheme 1


29

Page 31 of 37

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

Langmuir

Figure 1 263x136mm (93 x 93 DPI)


Langmuir

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

Figure 2 199x292mm (150 x 150 DPI)


Page 32 of 37

Page 33 of 37

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

Langmuir

Figure 3 202x293mm (150 x 150 DPI)


Langmuir

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

Figure 4 313x117mm (150 x 150 DPI)


Page 34 of 37

Page 35 of 37

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

Langmuir

Figure 5 318x127mm (150 x 150 DPI)


Langmuir

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

Figure 6 156x123mm (150 x 150 DPI)


Page 36 of 37

Page 37 of 37

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

Langmuir

Figure 7 157x123mm (150 x 150 DPI)


Xylanase Immobilized on Novel Multifunctional Hyperbranched

Recommend Documents