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Comparing immobilized cellulase activity in a magnetic threephase fluidized bed reactor under three types of magnetic field Jun Cui, Lin Li, Lingmei Kou, Hui Rong, Bing Li, and Xia Zhang Ind. Eng. Chem. Res., Just Accepted Manuscript • DOI: 10.1021/acs.iecr.8b02195 • Publication Date (Web): 19 Jul 2018 Downloaded from http://pubs.acs.org on July 29, 2018

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Industrial & Engineering Chemistry Research

Comparing immobilized cellulase activity in a magnetic three-phase fluidized bed reactor under three types of magnetic field

Jun Cui a, Lin Li a, b, c, Lingmei Kou a, Hui Rong d, Bing Li a, c, *`, Xia Zhang a, c, * a

School of Food Science and Engineering, South China University of Technology, 381 Wushan Road,

Guangzhou, 510640, China b

School of Chemical Engineering and Energy Technology, Dongguan University of Technology, College

Road 1, Dongguan, 523808, China c

Guangdong Province Key Laboratory for Green Processing of Natural Products and Product Safety, 381

Wushan Road, Guangzhou, 510640, China d

Guangzhou Entry-Exit Inspection & Quarantine Bureau of the People’s Republic of China, Guangzhou

510623, China

* Corresponding author: Bing Li, E-mail: [email protected], Tel/Fax: +86 20 87113252 Affiliation address: School of Food Science and Engineering, South China University of Technology, 381 Wushan Road, Guangzhou, 510640, China. Xia Zhang, E-mail: [email protected], Tel/Fax: +86 20 87113252 Affiliation address: School of Food Science and Engineering, South China University of Technology, 381 Wushan Road, Guangzhou, 510640, China.

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Abstract A novel, magnetic three-phase fluidized bed reactor (MTFBR) was designed with magnetic immobilized cellulase (MIC) as the biocatalyst for preparing chitooligosaccharides from chitosan. The MIC showed higher enzyme activity in a pulse magnetic field (PMF) than that in a steady magnetic field (SMF) or alternating magnetic field (AMF) under certain operating parameters. The application of a magnetic field increased the maximum reaction rate (Vmax) and Michaelis constant (Km), and the reaction rates of the MIC-catalysed reaction in PMF and SMF at high concentration of chitosan solution (Cs, 5 < Cs < 20 mg/mL) increased as the intensity of the magnetic field increased. The concentrations of chitosan pentamer and hexamer produced were higher in the PMF than that in the SMF and AMF, and the application of a magnetic field greatly reduced (by as much as 37.5%) the biocatalytic reaction time required to reach the maximum concentration of the desired chitooligosaccharides. These results suggest that the customized MTFBR supplemented with MIC is a reusable and effective piece of biocatalytic equipment, indicating that it has potential applications for large industrial-scale chitooligosaccharide syntheses. Keywords: three-phase fluidized bed reactor, magnetic field, magnetic immobilized cellulase, activity, chitooligosaccharides.

2


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1. Introduction Chitooligosaccharides are the degraded products of chitosan with molecular weights (MWs) of less than 100 glucosamine units, i.e., MW ≈ 16 kDa. Chitooligosaccharides exhibit excellent bioactivities such as anti-bacterial, hydrating, anti-cancer and immune enhancing effects

1–4

. In addition, given their

biodegradability and biocompatibility, the potential applications of chitooligosaccharides in the food and pharmaceutical industries are attractive

5,6

. Generally, chitooligosaccharides are prepared by the degradation

of chitosan using acid hydrolysis, physical treatment or enzymatic hydrolysis

7,8

. The enzymatic method

using cellulase is an environmentally friendly and efficient way to degrade chitosan 9. However, the use of enzymatic hydrolysis in large-scale industrial applications is limited because of the high costs and relative instability of the enzymes. Enzyme immobilization techniques can effectively improve the stability and reusability of enzymes, thus 10–12

lowering the cost and enhancing the practicality of enzymatic reactions for large-scale production

.

Enzyme immobilization has advantages over the use of free enzymes for improving the recovery rate of the biocatalyst during batch treatment in a stir tank reactor or in continuous processes in a fluidized bed

13

.

However, in terms of conventional fluidized bed reactors (FBRs), if the density of the immobilized enzymes is similar to that of the fluidized liquid, the enzyme could be carried away by the fluidized liquid, thus limiting the operating range and applicability. In addition, fluidization and stabilization problems may appear in FBRs when small immobilized enzymes are used. However, these problems could be solved by applying a magnetic field to an FBR containing magnetically immobilized enzymes 14,15. Several reports have presented magnetic fluidized bed reactors (MFBRs) as novel and efficient combinations of fluidization and magnetic technology for performing biological reactions

16,17

. By

introducing external magnetic fields and enzymes immobilized on magnetic carriers, the catalytic processing can remain stable under certain fluidization velocities as a result of the combination of the drag force and magnetic force 18. Once the FBR is equipped with a magnetic field generator, the hydrodynamic properties of 3



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the bed can be tuned by altering the intensity of the magnetic field and the properties of the magnetic particles 19. Besides, the presented flow regimes can be altered in different patterns by changing the applying order of magnetic field, such as packed bed, stable bed and fluidized regime for magnetization first mode, while fluidized bed, stabilized bed and frozen bed for magnetization last mode20. Among those regimes, magnetic field intensity played an important role in affecting the minimum fluidization velocities and particle agglomerations of magnetically stabilized systems, and the variations in the hydrodynamic behavior could therefore resulted in different biocatalytic efficiency21,22. Previous studies have reported the activity and kinetic parameters of immobilized glucoamylase in a magnetically stabilized FBR under different magnetic field intensities and substrate flow rates. Using a weak magnetic field intensity avoided the problems associated with diffusion and channelling that resulted from particle agglomeration and bed contraction 14. In addition, the activity and secondary conformation of cellulase can be altered by various intensities of static magnetic fields 23. However, to date, studies on the use of magnetic fields in catalytic process have mainly focused on steady magnetic fields (SMFs), whereas the biotechnologically promising idea of using an alternating magnetic field (AMF) or a pulse magnetic field (PMF) remained unexplored. In this study, our objectives were to prepare chitooligosaccharides from chitosan by hydrolysis using cellulases immobilized on magnetic microspheres in a magnetic three-phase (solid, liquid and gas phase) fluidized bed reactor (MTFBR), and to compare the activities of magnetic immobilized cellulase (MIC) in the MTFBR under different types of magnetic fields (SMF, AMF and PMF) and different parameters for the fluidization process. In addition, the kinetic parameters of the MIC under a low concentration of chitosan and the reaction rates in the MTFBR using high concentrations of chitosan were analyzed and compared with the same

parameters

of

the

analogous

non-magnetic

system.

For

further

industrial

applications

chitooligosacchrides preparation, we also examined the distribution of degraded products and reaction efficiency using this MTFBR. 4


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2. Materials and methods 2.1 Materials Chitosan (degree of deacetylation, 90%) and cellulase were provided by BoAo Biotechnology Co., Ltd (Shanghai, China). Ferriferrous oxide (Fe3O4) was purchased from Shanghai Engineering Group Factory (Shanghai, China). Span-80, Glutaraldehyde, formaldehyde, sodium acetate trihydrate, butanol, acetic acid, anhydrous ethanol and petroleum ether (boiling range: 60 - 90°C) were purchased from Damao Chemical Reagent Factory (Tianjin, China) and were of analytical grade. Liquid paraffin was purchased from Guangzhou Guanghua Sci-Tech Co., Ltd (Guangzhou, China).The other chemical reagents were of analytical grade. 2.2 Preparation of magnetic chitosan microspheres (MCMs) MCMs were prepared according to a previously described method with slight modifications 24. Typically, 0.5 g of the magnetic material (Fe3O4) was thoroughly mixed in a chitosan solution (4.5%; w/v), the resulted solution was then added with 2 mL of Span-80, 4 mL of butanol and 50 mL of liquid paraffin. The resulting solution was stirred with a mechanical stirrer at 2000 rpm for 30 min and then heated to 60°C before 2 mL of formaldehyde was added. The resulted dispersion was then heated again to 80°C, and 1.5 mL of glutaraldehyde was added, followed by stirring for an additional 1 hour. At the end of this period, the mixture of products was successively washed with petroleum ether, anhydrous ethanol and distilled water. After washing, the MCMs were collected using a magnet and then dried in an oven at 40°C for 24 h. The MCMs were stored in a vacuum desiccator until further use. 2.3 Immobilization of cellulase The immobilization procedures were conducted under the previously reported optimum conditions with slight modifications 14. MCMs (1.0 g) with an average diameter of 125 µm were allowed to swell in a sodium acetate acetic acid buffer solution (0.05 M, pH =5.0) for 10 h. After filtering the solution, a specific amount 5



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of cellulase solution (1.0 mg/mL) was added, and the mixture was then agitated in a constant temperature oscillator (SHA-BA, Fuhua Instruments and Equipment Co., Ltd, China) at 30°C and 150 rpm to obtain the MIC. The supernatant was removed and the MIC was repeatedly washed with the same sodium acetate-acetic acid buffer solution (0.05 M, pH =5.0) until no protein was detected. The prepared MIC was stored at 4°C until subsequent use. 2.4 Activity determination of the MIC A cellulase activity unit is defined as the amount of reducing sugar produced per unit time (µmol/min/g; IU/g) at certain pH and temperature. In this experiment, 50 g of MIC was loaded in the MTFBR, and chitosan solution (20 mg/mL; pH =4.5) was used as substrate. The enzymatic hydrolysis was performed in this MTFBR under specific processing parameters (liquid velocity (Ul), gas velocity (Ug), magnetic field intensity (H), treatment temperature and treatment time). The activity of the MIC was measured by determining the amount of reducing sugar produced in the abovementioned hydrolysis reaction using the dinitrosalicylic acid (DNS) method 25. 2.5 Magnetic three-phase fluidized bed reactor (MTFBR) All experimental measurements were performed using a setup designed in-house (Figure 1). A three-phase fluidized bed reactor composed of a gas phase (fresh air), liquid phase (chitosan) and solid phase (MIC) was developed. A magnetic field generator was designed as an accessory device to produce three types of magnetic fields: an SMF, AMF and PMF. A schematic drawing of the MTFBR is provided in Figure 1. The reactor mainly comprised a recirculation tank, an air compressor (OD1012, Longhailiba Universal Machine Co., Ltd, China), an FBR, an ultrafiltration unit (RO-40U, Yadong Resin Co., Ltd, China) and a production reservoir. The magnetic field was produced by using two Helmholtz solenoids (di=60 mm, do=140 mm, height= 100 mm) which were vertically arrayed along the column of the fluidized bed. The magnetic field intensity was adjusted by controlling the current passing through the Helmholtz solenoids by using an 6


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alternative current (AC) power supply. In addition, a range of accessories were matched to the power supply to produce the different magnetic fields. For the SMF, a silicon-controlled rectifier (HPW6S60B, Hao Pin Microelectronics Co., Ltd) was used to convert the AC to direct current (DC). For the AMF, a voltage transformer (5KVA, Jinxiang Welding Machine Factory, China) was used to transform the 220 V AC to the desired voltage. For the PMF, an electric filter was cascaded after the abovementioned voltage transformer and the passed current was split in half to generate a PMF by passing through the solenoids. The fluidized bed reactor was filled with 50 g of MIC, and the temperature of the substrate was controlled by a water bath. The compressed air was used as the carrier gas. A metering pump (BB05, Nikkiso Eiko, Japan) was used to control the minimum fluidization liquid velocity and circulate the substrate solution through the MTFBR. When the product passed through the ultrafiltration (UF) unit with molecular weight cut-off (MWCO) of < 10000, the undegraded products were trapped and transferred to the recirculation tank, while the desired degraded products flowed into the production reservoir. The activity of the MIC in the MTFBR was calculated by the method described in section 2.4.

Figure 1. Schematic diagram of the magnetic three phase fluidized bed reactor for chitosan hydrolysis

7



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2.6 Effects of processing parameters on the MIC activity in the MTFBR According to the abovementioned method (2.4), the treatment time was fixed at 30 min, and Ug and Ul were set at 50 mL/min and 20 mL/min, respectively. The MIC activity under the three different magnetic fields at 20°C and 50°C was measured as the H was adjusted to 0.0~5.0 kA/m (for SMF and AMF) or 0.0~1.5 kA/m (for PMF). Notably, according to the Biot-Savart-Laplace Law, the intensity of the magnetic field is positively correlated with the exciting current, and the adjustable range of the magnetic field intensity of PMF was 0 to 1.5 kA/m as a result of the split current. To determine the effect of the magnetic treatment time (0, 15, 30, 45, 60, 75, and 100 min) on the MIC activity, the temperature was set at 50°C, the magnetic field intensity was fixed at 1.5 kA/m for the three types of magnetic fields, and Ug and Ul were set at 50 mL/min and 20 mL/min, respectively. The effects of Ul (5, 10, 15, 20, 25, and 30 mL/min) on MIC activity were measured at 50°C, a Ug of 50 mL/min, a magnetic field intensity of 1.5 kA/m and a treatment time of 30 min. The effects of Ug (0, 30, 40, 50, 60, and 70 mL/min) on the MIC activity were measured at 50°C, a Ul of 20 mL/min, magnetic field intensity of 1.5 kA/m and a treatment time of 30 min. 2.7 Determination of the maximum reaction rate (Vmax) and Michaelis constant (Km) of the MIC Chitosan solutions at different concentrations (Cs of 1, 2, 3, 4, 5, 10, 15, and 20 mg/mL) were prepared. These solutions were used as substrates and pumped into the MTFBR by the metering pump (Ul= 20 mL/min) after 50 g of MIC had been loaded into the reactor. Ug was set at 30 mL/min and the treatment time was approximately 30 min for the different types of magnetic fields at 50°C. The degraded products of chitosan were trapped by the UF unit, and the reaction rate was calculated by determining the absorbance of the reducing sugars in the chitosan degradation products at 540 nm. The concentration of reducing sugar was measured by the DNS method as mentioned above. Lineweaver-Burk double-reciprocal plots were used to determine the Km and Vmax values by measuring the initial reaction rates of the MIC. 2.8 Determination of the chitosan degradation products in the MTFBR 8


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MIC (50 g) was transferred to the magnetic fluidized bed along with a specific concentration of chitosan in acetic acid under an SMF, an AMF or a PMF. The parameters for the enzymatic reaction were as follows: Ul = 20 mL/min, T = 50°C, and Ug = 30 mL/min. The degradation products (< 10000 kDa) were collected, and a UF unit (MWCO of 5000 kDa) was used to collect the smaller degradation products (< 5000 kDa). The final collected products were further fractionally precipitated to obtain three portions (A, B and C) with different degree of polymerization (DP, A: DP > 16; B: 8 < DP < 16; C: DP < 8) by using methanol/water (1:1 and 9:1, v/v) according to a previously reported method 26. The precipitated components were then collected by centrifugation at 5000 rpm for 30 min and vacuum-dried to prepare them for further use respectively. A precisely weighed 50 mg portion of the C component was transferred to a volumetric flask and dissolved in 10 mL of distilled water. The solution (5 mg/mL) was then centrifuged at 6000 rpm for 15 min, and the supernatant was collected and filtered through a 0.45-µm filter. The chitooligosaccharides in the filtered chitosan solution were identified and quantified by high-performance liquid chromatography (HPLC) on a Waters Spherisorb NH2-60 column (250 × 4.6 mm, Waters Co. USA). The sample was eluted with a 60 : 40 (v/v) mixture of CH3CN/H2O as the mobile phase at a flow rate of 0.8 mL/min 27. A chitooligosaccharide standard mixture containing d-glucosamine (GlcN) and chitosan dimer, trimer, tetramer, pentamer and hexamer was used as the internal standard. Identification and quantification of the degradation products were performed based on the peak areas and retention times in the HPLC profile and compared to the standard curve obtained from the chitooligosaccharides mixed-standard. 3 Results and discussion 3.1 Effects of processing parameters on MIC activity in the MTFBR 3.1.1 Magnetic field intensity

9



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Figure 2. Effects of magnetic field intensity on the MIC activities under different types of magnetic fields at 20°C (a) and 50°C (b). (Cs: 20 mg/mL, Ul: 20 mL/min, Ug: 50 mL/min, treatment time: 30min).

The variations in the MIC activities with different field intensities in the presence of an SMF, AMF or PMF at different temperatures are illustrated in Figure 2. The MIC activities were higher in a PMF than that in an SMF and AMF at the same magnetic field intensities. The MIC activity under the PMF at 20°C did not vary linearly and reached its highest value (10.01 UI/g) at 0.5 kA/m, followed by a decrease to 6.12 UI/g as the magnetic field intensity increased to 1.5 kA/m (Figure 2a). The initial increase in the activity of MIC could be due to the application of magnetic field which increased the contact between the MIC and the substrate. However, as the PMF intensity increased further, the magnetic agglomeration occurred as a result of the combination of the increasing magnetic field and the high viscosity of the substrate (11.4 mPa·s) at 20°C, leading to the decrease in MIC activity. As shown in Figure 2 (b), at 50°C, the MIC activity rapidly and monotonously increased as the magnetic field intensity increased, reaching the highest value (23.29 UI/g) at 1.5 kA/m. Due to the split current passing through the solenoids, the generated PMF imposed an intermittent magnetic force on the MIC. The periodic motion increased the contact time between the MIC and substrate since the impact of solution viscosity (1.94 mPa·s) at 50°C was not dominant. This behaviour was explained by previous study in which oscillation was found to enhance the molecular movement in a magnetic fluidized bed and overcome the diffusion resistance between MIC and products, thus increasing the mass-transfer efficiency 28,29. 10


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Although MIC in the SMF and AMF exhibited higher activities at 50°C than that at 20°C for intensities ranging from 0 to 4.0 kA/m, the change trends of MIC activity in the SMF and AMF varied differently. At 20°C, the MIC activities under the SMF and AMF rapidly decreased (0 to 0.5 kA/m) (Figure 2a), consistent with a previous study

16

that reported a weak magnetic field was not being sufficient to vibrate the

immobilized enzyme, and consequently, decreasing the contact time between the MIC and substrate. When H was > 0.5 kA/m, the MIC activity under the AMF showed a decreasing trend with increasing intensity but was steady under the SMF, which may be due to the alteration of the conformation of MIC and therefore affecting the active site of enzymes

30,31

. By contrast, at 20°C and in the chitosan solution with higher

viscosity, the reciprocating vibration of MIC particles caused by the AMF would facilitate the magnetic agglomeration, resulting in poor contact between the substrate and enzymes compared to the SMF. As the temperature increased to 50°C, the MIC activities under the SMF and AMF both gradually increased as the magnetic field intensity increased from 0 to 2.5 kA/m and then decreased dramatically as magnetic field intensity further increased to 5.0 kA/m. The low viscosity of the substrate at 50°C enabled higher mass-transfer efficiency

32

. When enzymes are exposed to a magnetic field, changes in their activities are

closely related to the magnetic field intensity. A previous study demonstrated that the activity of cellulase increased under 0 to 0.30 T magnetic fields, but decreased under a 0.45 T magnetic field due to magnetic agglomeration 23. 3.1.2 Treatment time

11



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Figure 3. Effects of treatment time on the MIC activities under different types of magnetic fields. (H: 1.5 kA/m, Cs: 20 mg/mL, Ul: 20 mL/min, Ug: 50 mL/min, T: 50°C).

When MIC was used as a biocatalyst in the MTFBR, as shown in Figure 3, the MIC activities exhibited similar trends under the SMF, AMF and PMF with prolonged treatment time, reaching a maximum at 30 min and them slowly decreasing with time. The initial decreases in the MIC activities under the SMF and AMF indicate that a balance time was necessary for MIC particles to reach stabilization under certain flow rates and magnetic field forces. However, the three dimensional structure of enzymes is flexible in nature, and when a magnetic field is exerted, in most cases the exposure of enzyme active sites would increase in such a way that more fitted the substrate. For all three types of magnetic fields, the MIC activities decreased with longer exposure time, mainly due to the accumulation of degradation products in the microenvironment of MIC, consistent with a previous report 23. These results suggested the PMF was favorable for improving MIC activity and that a moderate time (30 min) was optimal to achieve the maximum activity. 3.1.3 Effect of liquid and gas velocities on MIC activity

12


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Figure 4. Effects of Ug (a) and Ul (b) on the MIC activities under different types of magnetic fields (a) (H: 1.5 kA/m, Cs: 20 mg/mL, Ug: 50 mL/min, treatment time: 30 min, T: 50°C). (b) (H: 1.5 kA/m, Cs: 20 mg/mL, Ul: 20 mL/min, treatment time: 30 min, T: 50°C).

Ug and Ul substantially influence the amount of contact between the enzyme and substrate and also impact the enzyme activity. In general, the MIC activities under the three magnetic fields increased over time, and the highest enzyme activities were reached at Ul =20 mL/min (Figure 4a) or at Ug =50 mL/min (Figure 4b). The MIC activities with different Ug and Ul values were generally higher in the PMF than those under the SMF and AMF. Using viscous solutions as the liquid phase could result in low turbulence density

33

,

whereas higher Ug and Ul tend to be beneficial for reducing the boundary layer and improving the transfer of degradation products from the MIC microenvironment, thus resulting in an improvement of the MIC activities. When Ul > 20 mL/min or Ug > 50 mL/min, an exaggerated drag force on the MIC particles was generated, resulting in particle agglomeration that could induce mass transfer resistance, thus decreasing MIC activity. Moreover, the introduction of an external magnetic field force stabilized the reaction bed and decreased the liquid-phase dispersion, whereas a relatively high flow rate has been suggested to cause less stabilization in the magnetically stabilized fluidized bed due to the intensifying disturbance 34. 3.2 Kinetic parameters of MIC in the MTFBR at low substrate concentrations (0 < Cs < 5 mg/mL) A two-step mechanism for the cellulase-catalysed hydrolysis of chitosan has been reported previously 35. Our current understanding of the hydrolysis of chitosan in conjunction with a simple rate equation and reaction processing can be simply expressed by the following equation: 13



S+E

[ES]

Page 14 of 29

P+E (1)

This equation describes the initial binding of cellulase (E) and chitosan (S) to form an active enzyme-substrate complex (ES), followed by the depolymerization of this complex to further form product P and release enzyme from the substrate. In this simplified mechanism, the cellulase system is treated as a single enzyme in the initial stages of depolymerization The Michaelis-Menten constants (Km) and maximum reaction rates (Vmax) of the MIC-catalysed reaction were measured under each type of magnetic field at different magnetic field intensities using the Michaelis– Menten equation:

V =

Vmax Cs

(2)

Km +Cs

where V is the reaction rate, Cs is the substrate concentration, Vmax is the maximum reaction rate and Km is the substrate concentration at the half-maximal rate. Lineweaver-Burk double-reciprocal plots were used to investigate the MIC kinetic parameters, Km and

Vmax in different magnetic fields (Figure 5). In general, the pattern of chitosan degradation by the MIC was not actually affected by the application of the magnetic field as indicated by the linear relationship presented in the Lineweaver-Burk double-reciprocal plots. In addition, the regression curves in the absence of a magnetic field had the highest Y-intercept (Figure 5), suggesting that the application of a magnetic field increased Vmax. Similar results were reported for immobilized catalase exposed to a magnetic field

36

.

Immobilization of cellulase can provide a rigid structure that will enhance the stability of MIC and provide an expanded surface for enzymes to bind, possibly because the structural orientations of MIC will expose more available active site under magnetic fields

37,38

. In addition, the higher X-intercept of the regression

curve in the absence of a magnetic field indicates a lower Km and implies that MIC has higher affinity for the substrate in a non-magnetic system than in the MTFBR, consistent with previous reports 39 .

14


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Figure 5. Lineweaver-Burk double-reciprocal plots with chitosan as the substrate of MIC in an (a) SMF, an (b) AMF, and a (c) PMF. (Ul: 20 mL/min, Ug: 30 mL/min, treatment time: 30 min, T: 50°C).

As shown in Table 1, increasing the magnetic field intensity of the SMF significantly increased the Vmax of the MIC-catalysed reaction from 2.7601 to 9.4607 mg·min-1. Similar results for the trends of the Km and Vmax values of cellulase in a static magnetic field were reported previously 23. For the AMF, as the magnetic field intensity increased to H = 0.5 kA/m, the Vmax and Km of the MIC-catalysed reaction were 3 times greater than those of the reaction in the absence of a magnetic field. Compared with SMF and AMF, MIC exhibited a higher Km value at H =1.5 kA/m in the PMF, indicating a lower affinity for the substrate. At low substrate concentration, viscosity has little effect on the motions of MIC particles, and the intermittent magnetic force generated by the PMF may decrease the contact time between the MIC and substrate compared to the continuous magnetic force generated by the SMF and AMF. In general, the Km and Vmax values of the MIC-catalysed reaction increased as the magnetic field intensity increased for all three types of fields. The 15



Page 16 of 29

increased Km value could be explained by the changes in the conformation of the active sites that resulted from the increase in the magnetic field intensity. Moreover, the MIC would be subjected to the superimposition of the induced and external magnetic fields, which would enhance the interactions between particles and lead to the magnetic agglomeration, with an increase in the Km 40. Table 1 Kinetic constants of the MIC-catalysed reaction in a steady magnetic field, an alternating magnetic field and a pulse magnetic field Magnetic field intensity

Km

Vmax

R2

Vmax/Km

2.7601

0.9295

1.111

4.9199

5.5249

0.9632

1.250

H＝1.5

5.7525

8.1699

0.9936

1.422

H＝2.5

7.4560

9.4607

0.9859

1.269

H＝0.0

2.4839

2.7601

0.9295

1.111

H＝0.5

3.2125

3.7230

0.9868

1.159

H＝1.5

4.1393

4.5085

0.9617

1.089

H＝2.5

7.5652

7.4184

0.9858

0.981

H＝0.0

2.4839

2.7601

0.9295

1.111

H＝0.5

3.7916

3.5149

0.9655

0.927

H＝1.0

5.3969

4.8054

0.9885

0.890

H＝1.5

6.4507

6.0827

0.9800

0.943

-1

mg·mL

mg·min-1

H＝0.0

2.4839

H＝0.5

kA/m

SMF

AMF

PMF

3.3 Reaction rate of the MIC-catalysed reaction in the MTFBR with high substrate concentrations (5 < Cs < 20 mg/mL) Usually, the catalytic reaction at a high concentration of substrate is quite different from that at a low concentration of substrate due to different diffusion limitations

41,42

. However, the situation becomes more

complex when external magnetic fields are introduced as part of this reactor. The dependence of the variation in reaction rate of the MIC-catalysed reaction on the different magnetic fields at high substrate concentrations (5 < Cs < 20 mg/mL) is presented in Figure 6. The reaction rates of the MIC-catalysed reaction were significantly affected by both the type and intensity of the magnetic field. The reaction rates of the MIC-catalysed reaction under the SMF increased as the magnetic field intensity 16


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increased (Figure 6a).By contrast, the decrease in the reaction rate under H = 0.5 kA/m was mainly due to the weak intensity of the magnetic field, which was unable to increase the motions of MIC in the substrate. For the AMF, the reaction rates of the MIC-catalysed reaction present a decreasing trend as Cs increased (Figure 6b). Notably, when Cs increased from 5 to 15 mg/mL, the reaction rates showed a decrease trend as the magnetic field intensity increased, in contrast to the profiles in SMF. However, when Cs further increased to 20 mg/mL, the reaction rate of the MIC-catalysed reaction in AMF under H = 0.5 kA/m decreased dramatically and reached the minimum, reflecting limited motions of MIC in a viscous substrate as described above. In addition, the reaction rate profiles in the AMF (Cs = 20 mg/mL) also indicated that a moderate intensity of magnetic field (H = 1.5 kA/m) could result in a higher reaction rate.

Figure 6. Reaction rate of the MIC-catalysed reaction in the SMF (a), AMF (b) and PMF (c) with different magnetic field intensities. (Ul: 20 mL/min, Ug: 30 mL/min, treatment time: 30 min, T: 50°C).

As shown in Figure 6c, the reaction rates of the MIC-catalysed reaction in the PMF increased initially and then decreased slowly as Cs further increased to 20 mg/mL. In addition, the reaction rate at 1.5 kA/m under the PMF was higher than that at other intensities at the same Cs. This result corresponds to the higher 17



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MIC activities under the PMF at fixed Ug and Ul values than those observed under the SMF and AMF at 1.5 kA/m (Figure 3). This activation by the PMF could contribute to the formation of more active sites or enlarge existing sites, resulting in higher binding ratios in high Cs reaction mixtures and thus increasing the reaction rate 43. Note that, compared with that in SMF and AMF, the overall higher reaction rates in the PMF at 1.5 kA/m indicates that this value of magnetic field intensity in a PMF has general applicability over a wide range of substrate concentration. These results indicated that a higher magnetic field intensity applied in the SMF and PMF was optimal for the MIC-catalysed reaction, whereas a moderate magnetic field intensity was appropriate for the MIC-catalysed reaction in an AMF. The difference in the optimal magnetic field intensity is partially due to the variance in the different function patterns of the magnetic fields. In addition, the orientation of the magnetic fields can also influence the optimal magnetic field, as it was proved that an axial magnetic field tended to result in more stable fluidization at higher flow velocity compared to a transverse one under the same intensity 44. 3.4 Distribution of chitooligosaccharides in the MTFBR To further evaluate the efficiency of this apparatus, we chose chitosan pentamer and hexamer (C(5+6)) as the target chitooligosaccharide products to investigate the process of degradation of chitosan in the MTFBR. These molecules are not only common degradation products but also possess potent physiological activities, such as anti-microbial functions and anti-inflammatory effects45.

18


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Figure 7. Effects of the types of magnetic fields on the proportions of chitooligosaccharides (pentamer and hexamer) at different chitosan concentrations (a) and treatment times (b). SMF, AMF, PMF and NMF stand for steady magnetic field, alternating magnetic field, pulse magnetic field and non-magnetic field, respectively. (a) (H: 1.5 kA/m, Ul: 20 mL/min, Ug: 30 mL/min, treatment time: 150 min, T: 50°C). (b) (H: 1.5 kA/m, Ul: 20 mL/min, Ug: 30 mL/min, Cs = 20 mg/mL, T: 50°C).

As shown in Figure 7a, the proportions of C(5+6) in different MTFBRs all increased as Cs increased. In addition, the rate of the increase in the proportion of C(5+6) was slower at Cs < 5 mg/mL than at 5 < Cs < 10 mg/mL, potentially indicating that a higher Cs provides more enzymatic reaction substrates. Given the intermittent motions of the MIC due to the PMF, a slightly higher concentration of C(5+6) was observed under the PMF as Cs increased to 30 mg/mL, coinciding with the higher MIC activity under the PMF in Fig 2b and the higher reaction rate under the PMF in Fig 6c. The effects of treatment time on the proportion of C(5+6) degradation products under different magnetic fields are illustrated in Figure 7b. The trends in C(5+6) generated under different magnetic fields were indentical with nonmonotonic characteristics, and the maximum concentrations (7.52 mg/mL under the PMF) were reached at 150 min. However, the control reactions (dashed line in Figure 7b, no applied magnetic field) presented slower reaction rates; 240 min was required to reach the maximum concentration of C(5+6). Regardless of the higher C(5+6) concentration in the control, the introduction of a magnetic field significantly decreased the treatment time for the MIC-catalysed degradation of chitosan into chitosan pentamers and hexamers. In general, the immobilization of enzymes can impart a rigid structure that protects the active site 19



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of enzymes from thermal or pH deterioration, but this increased rigidity often decreases their affinity for the substrate

29,46

. Because ferroferric oxide makes the immobilized carrier a paramagnetic material, MIC can

easily form clusters, reducing the flexibility of the enzyme and thus decreasing the exposure of active sites 47. For the non-magnetic field bed reactor, high initial chitosan concentrations decreased the mass-transfer efficiency because the degradation products could not be transferred in a timely manner, and products accumulated in the microenvironment of the MICs, thus impeding the enzymatic reaction. Compared to that under the non-magnetic field, the reaction rate of MIC was greatly improved under the magnetic field, as mentioned above (Table 1). The immobilization processing, which provides a rigid carrier for MIC, ensures that the magnetic field has little influence on the secondary structure of the enzyme

46

.A

previous study noted that the reaction rate of Ca+-binding protein may be influenced by single or multiple magnetic fields as a result of transitions between vibrational levels in electronic energy terms of the Ca2+-protein complex

48

. Therefore, the existing calcium ions in cellulase may be influenced by magnetic

fields and cause alterations of flexibility or three-dimensional structure of cellulase, thus resulting in variations of the MIC-catalysed reaction rate. Moreover, the contact between the MIC and the substrate was improved by the introduction of a magnetic field, in combination with other forces in the MTFBR. This improved contact greatly enhanced the transfer of the degradation products from the microenvironment of MIC to the solution, resulting in a decrease in the time needed to reach the maximum concentration of C(5+6), and significantly improving the reaction efficiency. From a productivity perspective, this MTFBR has promising applications in bio-industry. 4. Conclusion In the present work, MIC activity increased as the magnetic field intensity increased to 1.5 kA/m at 50°C, and MIC activity was higher under the PMF than that under the SMF and AMF. Further increases in the magnetic field intensity decreased MIC activity. For all three magnetic fields, MIC activities were maximal at 20


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an Ug of 50 mL/min and an Ul of 20 mL/min. Increases in treatment time increased the MIC activities in magnetic fields in the first 30 min. At Cs < 5 mg/mL, the application of a magnetic field increased Km, indicating that the enzyme affinity decreased with increasing magnetic field intensity. The reaction rate of the MIC-catalysed reaction in PMF and SMF at high Cs (5 < Cs < 20 mg/mL) increased as the intensity of the magnetic field increased. When magnetic fields were applied, the proportion of the degraded chosen target products of chitosan pentamer and hexamer (C(5+6)) increased with the chitosan concentration and reached the maximum more quick than that without magnetic field treatment. The results obtained in this study reveal that the operating parameters of the MTFBR with different types of magnetic fields might impact the activity of the MIC in different ways. This highly effective and environmentally friendly apparatus offers a useful reference for the industrial application of MTFBRs for the production of chitooligosaccharides. 5. Associated Content Supporting Information Available: **[Optical micrograph of MCMs; changes of minimum fluidized liquid velocity in SMF with different Ug; effect of absorptive time on the activity of MIC; effect of amounts of cellulase used in the absorptive process on the activity of MIC]** 6. Acknowledgements This work was financially supported by the National Key R & D Program of China (no. 2016YFD0400303); the Natural Science Foundation of Guangdong Province (no. 2017A030311021); and the Guangdong Province R & D Program (2012B020312002).

21



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