α-Glucosidase immobilization on chitosan-enriched magnetic composites for enzyme inhibitors screening

Accepted Manuscript Title: ␣-Glucosidase immobilization on chitosan-enriched magnetic composites for enzyme inhibitors screening Authors: Dong-Mei Liu, Juan Chen, Yan-Ping Shi PII: DOI: Reference:

S0141-8130(17)31528-3 http://dx.doi.org/doi:10.1016/j.ijbiomac.2017.07.045 BIOMAC 7853

To appear in:

International Journal of Biological Macromolecules

Received date: Revised date: Accepted date:

27-4-2017 29-6-2017 7-7-2017

Please cite this article as: Dong-Mei Liu, Juan Chen, Yan-Ping Shi, ␣Glucosidase immobilization on chitosan-enriched magnetic composites for enzyme inhibitors screening, International Journal of Biological Macromoleculeshttp://dx.doi.org/10.1016/j.ijbiomac.2017.07.045 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

α-Glucosidase immobilization on chitosan-enriched magnetic composites for enzyme inhibitors screening Dong-Mei Liu1, 2, Juan Chen, Yan-Ping Shi1 1

CAS Key Laboratory of Chemistry of Northwestern Plant Resources and Key

Laboratory for Natural Medicine of Gansu Province, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Lanzhou 730000, People’s Republic of China 2

University of Chinese Academy of Sciences, Chinese Academy of Sciences, Beijing

100049, P. R. China



Correspondence: Juan Chen, and Yan-Ping Shi; Tel.: 86-931-4968208; fax: 86-931-4968094; E-mail: [email protected] (J. Chen), [email protected] (Y. -P. Shi).

Highlights 

A new kind of magnetic chitosan composites were prepared by the simple embedding method.



The immobilized amount of α-Glu was improved as the high content of chitosan.



Immobilized α-Glu showed a broader working pH and temperature range.



The reusability and storage stability of α-Glu were improved after immobilization.



α-Glu inhibitors were screened from 11 TCMs and 9 vegetables.

Abstract In the present study, the chitosan-enriched magnetic composites (MCCs) were prepared by a novel and simple embedding method for the immobilization of αglucosidase (α-Glu). The immobilized α-Glu could be easily separated from the 1

reaction mixture under an external magnetic field owing to the magnetic support. With the MCCs-immobilized α-Glu, enzyme activity and stability were studied, and enzyme inhibitors were screened from traditional Chinese medicines (TCMs) and vegetables combined with capillary electrophoresis (CE). The MCCs-immobilized αGlu exhibited enhanced pH and temperature tolerance with unchanged optimum pH and temperature of 4.0 and 60 oC comparing with free α-Glu. Reusability of the immobilized α-Glu was significantly improved after immobilization, and it retained 62.2% of its initial activity after 10 repeated cycles. Immobilized α-Glu also showed improved storage stability (84.3 ± 1.2% after 35 days of storage at 4 oC). The kinetic parameter Km for immobilized α-Glu was calculated to be 0.81 mM and the affinity of enzyme towards its substrate was reduced after immobilization. Finally, immobilized α-Glu was used to screen enzyme inhibitors from the extracts of TCMs and vegetables. The enhanced pH and temperature tolerance, improved reusability and storage stability of MCCs-immobilized α-Glu make it a promising candidate for biotechnological applications. Keywords: Chitosan-enriched magnetic composites; Immobilization; α-Glucosidase; Kinetic study; Inhibitor screening 1. Introduction In enzyme assays, enzymes are usually immobilized onto solid supports to obtain immobilized enzymes owing to their superior advantages. After immobilization, enzymes are not easy to be contaminated and less labor are needed in the process. 2

Also, the hyperactivity, process control, catalytic process, functional efficacy, reusability and stability are improved [1]. In addition, enzymes are more resistant to environmental changes after immobilization, such as pH and temperature [2, 3]. Owing to their advantages stated above, immobilized enzymes have been widely used in various applications, such as biosensors [4], pharmaceutical industry [5], food industry [6], wastewater [7], textile industry [8], and biocatalytic process [9] etc. There have been many reviews about the immobilization methods, application fields, immobilization supports and properties of the immobilized enzymes [1, 10-12]. Besides nanomaterials, low cost and bio-friendly supporting polymers such as agar-agar, polyacrylamide, gelatin, Ca-alginate and chitosan have played a significant role as carrier materials for enzymes immobilization [3, 13-14]. Chitosan is considered to be an attractive support material for enzymes immobilization owing to its excellent biocompatibility and biodegradability. For examples, chitosan beads have been used to immobilize laccase [15], catalase [16], horseradish peroxidase [2], manganese peroxidase [17] and so forth. In addition, chitosan can also be used as modifier to ensure that enzymes can be immobilized onto other supports, such as hollow fiber membranes [18], SiO2 nanoparticles [19], carbon nanotubes [20], gold nanoparticles [21], cobalt oxide nanoparticles [22], and Fe3O4 nanoparticles [23] etc. Nanoparticles were found to be very attractive for enzymes immobilization owing to their large specific surface area and stable surface [13]. However, their properties of small size would cause the inconvenient separation of the immobilized enzymes from the reaction mixture. Magnetic supports can guarantee the easy separation of 3

immobilized enzymes from the reaction mixture. Owing to the property of easy separation, magnetic materials have been widely used as supports for enzymes immobilization. In combination of chitosan and Fe3O4 nanoparticles, magnetic chitosan composites synthesized by different methods have been reported. For examples, chitosan-coated magnetic nanoparticles were prepared via an improved hydrothermal method in one step in the presence of chitosan [24], superparamagnetic chitosan microparticles were synthesized in situ by electrostatic droplets technique [25], magnetic chitosan beads were prepared by a common embedding method [26], magnetic chitosan nanoparticles were prepared by electrostatic interaction and crosslinking with sodium tripolyphosphate [27]. Combining the distinct properties of both chitosan and Fe3O4 nanoparticles, the magnetic chitosan composites could provide alternative supports for enzymes immobilization. In our previous work [28], the magnetic chitosan nanoparticles were synthesized for α-glucosidase (α-Glu) immobilization with satisfactory properties. However, the synthesis method is a little tedious and the amount of immobilized enzyme is unsatisfactory caused by the low content of chitosan modified on the magnetic nanoparticles. In the present work, a much more facile method was developed to synthesize a new kind of chitosan-enriched magnetic composites (MCCs) with high content of chitosan to immobilize α-Glu. By simply adding the mixture of Fe3O4 nanoparticles and chitosan solution to sodium hydroxide solution, MCCs were prepared. Then, by crosslinking with glutaraldehyde (GA), α-Glu was covalently crosslinked on MCCs. The factors influencing the immobilization process and 4

enzymatic reaction were investigated. Enzyme activity was studied with the substrate of p-nitrophenyl-α-D-glucopyranoside (pNPG). The property of inhibition activity of the immobilized α-Glu was validated with the known inhibitor, acarbose. Finally, inhibitory potencies of 11 traditional Chinese medicines (TCMs) and 9 vegetables were determined in the present study. 2. Materials and methods 2.1 Materials All reagents were of analytical grade and used without further purification. αGlucosidase (EC 3.2.1.20, α-Glu) from Aspergillus niger was purchased from Beijing Solarbio Science & Technology Co., Ltd (Beijing, China). p-Nitrophenyl-α-Dglucopyranoside (pNPG) and acarbose were obtained from J&K Chemical Co., Ltd (Shanghai, China). Ferric chloride hexahydrate (FeCl3·6H2O), anhydrous sodium acetate (NaAc), sodium dihydrogen phosphate (NaH2PO4), sodium tetraborate decahydrate (Na2B4O7·10H2O), hydrochloric acid (HCl), sodium hydroxide (NaOH) and glutaraldehyde (GA, 25% w/v) were obtained from Tianjin Kermel Chemical Reagent Co., Ltd (Tianjin, China). Chitosan (biochemical grade) was obtained from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Ethylene glycol, polyethylene glycol (PEG) and ethanol and acetic acid (HAc) were supplied by Li’anlong Bohua Pharmaceutical Chemical Co., Ltd (Tianjin, China). Ultra-pure water used throughout the experiment was produced by a Milli-Q water system (Shanghai Laikie Instrument Co., Ltd, China). All TCMs in Table 1 were purchased 5

from a local Hui Ren Tang drug store (Lanzhou, China) and all vegetables in Table 2 were purchased from a local supermarket. 2.2 Solutions All solutions were prepared with ultra-pure water (18 MΩ cm) and filtered through a 0.45 μm nylon membrane filter. - Phosphate buffer: phosphate buffer was prepared by dissolving certain amounts of NaH2PO4 in ultra-pure water with the concentration of 20 mM. Its pH value was adjusted to 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 7.4 and 8.0 with 1.0 M HCl or 1.0 M NaOH. - Background electrolyte (BGE): borate buffer was used as separation buffer and was prepared by dissolving certain amounts of Na2B4O7·10H2O in ultra-pure water with the concentration of 20 mM. Its pH was adjusted to 9.0 with 1.0 M HCl. - Stock solutions: the stock solutions of substrate (pNPG) with the concentration of 10 mM, inhibitor (acarbose) with the concentration of 1.0 mM and α-Glu with diverse concentrations were prepared in phosphate buffer. All the stock solutions were stored at -20 oC in aliquot and diluted with phosphate buffer before use each day. - Sample solutions: air-dried TCMs were ground into fine powder with a pulverizer and fresh vegetables were cleaned with ultra-pure water and homogenized with a blender. The ground TCMs (2.0 g) and homogenized fresh vegetables (2.0 g) were respectively extracted with 20 mL of 70% (v/v) ethanol-H2O in an ultrasonic cleaner for 2 h at a frequency of 70 kHz and a temperature of 30 oC. After centrifuging the

6

mixture, the supernate was condensed to dryness with rotary evaporator. Then the residues were dissolved in 2 mL of phosphate buffer and stored at 4 oC. 2.3 Apparatus The crystal structure of Fe3O4 was measured with an X’pert PRO X-ray diffraction (XRD, PANalytical B.V., Netherlands) with Ni-filtered Cu Kα radiation in the range of 2θ from 20o to 70o. Fourier-transform infrared (FT-IR) spectra of chitosan, Fe3O4, MCCs, GA-modified MCCs and MCCs-immobilized α-Glu were obtained with an IFS120HR FT-IR spectrometer (Bruker, Germany) with the wave numbers in the range of 500-4000 cm-1. The morphologies of Fe3O4 and MCCs were observed by a transmission electron microscope (TEM, FEITECNAI G2TF20, USA). 2.4 Analytical methods An Agilent 7100 CE system (Agilent Technologies, Waldbronn, Germany) equipped with a diode array detector (DAD) was used for CE analysis. All data analysis and instrumental control were performed via an Agilent CE Chemstation (Rev. B 04.02). Uncoated fused-silica capillaries of 50 μm i.d. × 33 cm (effective length of 24.5 cm) were purchased from Ruifeng Chromatographic Devices (Yongnian, Hebei, China). The cartridge temperature was set at 25 oC. Separations were performed by applying a constant voltage of 20 kV and detections were performed at 405 nm with a bandwidth of 30 nm. The capillary was pretreated with 1 M NaOH for 30 min and ultra pure water for 10 min. At the beginning of each day, the capillary was conditioned with 1 M NaOH for 10 min, ultra pure water for 5 min and BGE for 5 min. Between runs, 7

the capillary was rinsed respectively with 1 M NaOH, ultra pure water and BGE for 1 min. At the end of each day, the capillary was rinsed with 1 M NaOH for 5 min and ultra pure water for 10 min to ensure a good cleaning of the capillary inner wall. 2.5 Immobilization of α-Glu onto GA-modified MCCs Firstly, highly dispersed magnetic nanoparticles were synthesized by solvothermal method [29]. Briefly, 1.35 g of FeCl3·6H2O was fully dissolved in 40 mL of ethylene glycol, then 3.6 g of NaAc and 1.0 g of PEG were added into the above obtained solution and vigorously stirred for 30 min. The mixture was sealed in a teflon-lined stainless-steel autoclave with a capacity of 50 mL and maintained at 200 oC for 24 h. When cooled to room temperature, the black products were washed with ethanol for several times and dried in a vacuum drying oven at 60 oC. For the synthesis of MCCs, 15 mg of chitosan was dissolved in 5 mL of 2% (v/v) acetic acid solution, then by ultrasonication, 15 mg of Fe3O4 nanoparticles were dispersed in the chitosan solution prepared above. The above mixture was added drop by drop into 10 mL of 1.0 M NaOH with vigorous magnetic stirring to form MCCs. The MCCs were thoroughly washed with ultra-pure water to give a neutral pH and the supernate was discarded by magnetic decantation. High content of chitosan would collapse during the drying process and this was adverse to the redispersion and further modification of MCCs. So the drying process was avoided and the MCCs were redispersed in 5 mL of ultra-pure water for further modification.

8

GA was used as the cross linker for α-Glu immobilization. 1 mL of the homogenous MCCs dispersion was transferred into a glass vial, the supernate was firstly discarded by magnetic decantation and 1.8 mL of pH 7.4 phosphate buffer was added into the glass vial. By ultrasonication, the MCCs were dispersed in the phosphate buffer, then 0.2 mL of GA was added into the MCCs solution under vigorous shaking. Then, the mixture was shaken for a certain time at room temperature. The resultant GA-modified MCCs were separated magnetically, washed with ultra-pure water for several times, and re-dispersed in 1 mL of phosphate buffer with different pH values. For α-Glu immobilization, 1 mL of α-Glu solution with different concentrations ranging from 0.6 to 2.0 mg mL-1 (final concentration ranging from 0.3 to 1.0 mg mL1

) and the above obtained GA-modified MCCs dispersion were mixed. After shaking

for a certain time, the MCCs-immobilized α-Glu was collected, washed several times with phosphate buffer, re-dispersed in 2 mL of phosphate buffer and stored at 4 oC for further study. The whole procedure for α-Glu immobilization is shown in Fig. 1. 2.6 Activity study of free and immobilized α-Glu For activity study, pNPG was used as substrate and the production of p-nitrophenol (pNP) was measured by CE analysis. The peak area of pNP was presumed as the enzyme activity [30]. For free α-Glu activity study, the enzymatic reaction was carried out with a final volume of 30 μL containing 20 mM phosphate buffer (pH 4.0), 0.5 mM of pNPG and 9

70 μg mL-1 of α-Glu. The reaction mixture was incubated at 60 oC for 10 min. The enzymatic reaction was stopped by freezing and analyzed by CE. For immobilized α-Glu activity study, the re-dispersed MCCs-immobilized αGlu was ultrasonicated to obtain a homogenous dispersion. 15 μL of the homogenous dispersion was firstly transferred into a 1.0 mL Eppendorf tube, then, 15 μL of pNPG was added into the above Eppendorf tube. The enzymatic reaction was carried out with a final volume of 30 μL containing 20 mM phosphate buffer (pH 4.0) and 0.5 mM pNPG. The reaction mixture was incubated at 60 oC for 10 min. The enzymatic reaction was terminated by magnetically separating the immobilized α-Glu from the reaction mixture. The reaction mixture was then injected into CE for analysis. 2.7 Kinetic study and inhibition study The kinetic and inhibition study were performed according to Section 2.6 with some modifications. After the transfer of 15 μL of homogenous dispersion into the Eppendorf tube, 15 μL of pNPG with or without inhibitors was added. The final concentration of pNPG was varied from 0.2 to 1.2 mM. After magnetic separation with the immobilized α-Glu, the reaction mixture was analyzed by CE. 2.8 Reusability and storage stability of α-Glu For reusability study, the immobilized α-Glu was incubated with 0.5 mM pNPG at 60 o

C for 10 min, and the peak area of pNP was measured by CE. After the first run, the

immobilized α-Glu was collected from the reaction mixture by magnetic separation and washed three times with 20 mM phosphate buffer (pH 4.0) for the next run. For 10

storage stabilities study, both the free and immobilized α-Glu were stored in 20 mM phosphate buffer (pH 4.0) at 4 oC. The storage stabilities were tested by periodic measurement of residual enzyme activity during the storage period of 35 days. 2.9 Statistical analysis All experiments (e.g., immobilization, enzyme assays, reusability studies, storage stability studies, kinetic studies and inhibitors screening) were carried out in triplicate (n = 3). The average of triplicate measurements was calculated along with the standard deviation (SD). Data were statistically analyzed using Microsoft Excel software (Microsoft) and GraphPad Prism 5.0. 3. Results and discussion 3.1 Characterization Fig. 2(A) shows the XRD pattern of the Fe3O4 nanoparticles. The diffraction peaks at 2θ = 30.0o, 35.2o, 43.0o, 53.3o, 56.9o and 62.5o corresponded to the (220), (311), (400), (422), (511) and (440) crystalline planes of Fe3O4. The results were in well agreement with those found in the database of JCPDS (PDF No. 65-3107) [31]. The FT-IR spectra of chitosan, Fe3O4, MCCs, GA-modified MCCs and MCCsimmobilized α-Glu are compared in this section, and the results are shown in Fig. 2(B). The peaks at 580 cm-1 (curves a, b, d and e) corresponded to the Fe-O bonds and the peaks became less pronounced (curves b, d and e) with the surface modifications. The characteristic adsorption band of the bending vibration of N-H at 1595 cm-1 for chitosan (curve c) shifted to 1530 cm-1 (curve b) because of the interaction with Fe3O4 11

nanoparticles. Comparing the FT-IR spectra of MCCs (curve b) and GA-modified MCCs (curve d), the bending vibration of N-H at 1530 cm-1 disappeared and the imine band νas(C=N) at 1725 cm-1 appeared. The results indicated the successful modification of GA on MCCs [32]. The peak at 1530 cm-1 corresponding to the bending vibration of N-H appeared again on curve e as the immobilization of α-Glu molecule. The morphological features of Fe3O4 and MCCs were investigated by TEM analysis. Fig. 3(A) shows the highly dispersed Fe3O4 nanoparticles synthesized by solvothermal method. After modification with chitosan (Fig. 3(B)), Fe3O4 nanoparticles were embedded on the chitosan membrane formed in concentrated alkaline solution. As shown in the inset of Fig. 3(B), the synthesized MCCs were flocculent and loose in aqueous solution due to the high content and repulsion of chitosan, and the magnetic property still maintained although with high content of chitosan. 3.2 Effects of immobilization conditions on enzyme activity It is of great importance to optimize the immobilization conditions in order to obtain the maximal enzyme activity. Therefore, the immobilization conditions of enzyme concentration, crosslinking time, immobilization pH and immobilization time were investigated. 3.2.1 Effect of final enzyme concentration

12

For immobilization, the final enzyme concentration ranging from 0.3 to 1.0 mg mL-1 with other fixed factors (crosslinking time of 3 h, immobilization pH of 5.0, immobilization time of 2 h) was investigated. As shown in Fig. 4(A), the relative activity increased with the increase of enzyme concentration from 0.3 to 0.8 mg mL-1 and reached its maximal at the concentration of 0.8 mg mL-1. Then, the relative activity decreased slightly when further increasing the enzyme concentration. The phenomenon might be explained by the fact that the enzyme would overload on the support with a higher enzyme concentration which would hide the active site, resulting in a decrease in enzyme activity [15, 33]. Therefore, the final enzyme concentration of 0.8 mg mL-1 was chosen for immobilization. 3.2.2 Effect of crosslinking time In order to evaluate the effect of crosslinking time on enzyme activity, the crosslinking time ranging from 1 to 7 h with other fixed factors (enzyme concentration of 0.8 mg mL-1, immobilization pH of 5.0, immobilization time of 2 h) was examined. As shown in Fig. 4(B), the relative activity reached its maximal when the crosslinking time was 3 h. This might be explained by the fact that the amount of reactive GA crosslinked on the supports reached its maximal after crosslinking for 3 h. As the Schiff base reaction would conduct between the supports, when further increasing the crosslinking time, the relative activity decreased owing to the reduction of the reactive GA. Also, the too crowded enzyme molecule on the support would hide the active site [27, 34]. A second peak at 6 h might be caused by the further

13

modification of the internal surface of the flocculent and loose supports. The crosslinking time of 3 h was chosen. 3.2.3 Effect of immobilization pH For immobilization pH optimization, the immobilization was performed at various pH values ranging from 3.0 to 7.0 with other fixed parameters (enzyme concentration of 0.8 mg mL-1, crosslinking time of 3 h, immobilization time of 2 h). As shown in Fig. 4(C), the relative activity reached its maximum at pH 5.0. The lower relative activity at pH 3.0 and 4.0 could be explained by the fact that acidic pH values were unfavorable for the Schiff base formation between the amino group of α-Glu and the aldehyde group of GA. Although the Schiff base was easier to form at higher pH values, α-Glu would be inactivated, so the relative activity decreased when further increasing the pH value up to 7.0. Therefore, the immobilization pH of 5.0 was chosen as the optimal immobilization pH. 3.2.4 Effect of immobilization time In order to investigate the effect of immobilization time on enzyme activity, the immobilization was performed at varied time (1 to 6 h) with other fixed factors (enzyme concentration of 0.8 mg mL-1, crosslinking time of 3 h, immobilization pH of 5.0). As shown in Fig. 4(D), the relative activity reached its maximal after 2 h of immobilization. When increasing the immobilization time up to 5 h, the immobilized enzyme on the supports would aggregate and become too crowded. This would hide the active sites for substrate [34]. With longer immobilization time, the immobilized 14

enzyme amount increased by immobilizing on the internal surface of the flocculent and loose supports, so a second peak was found after 6 h of immobilization. The immobilization time of 2 h was chosen for enzyme immobilization. 3.3 The immobilized amount of α-Glu The amount of α-Glu immobilized on MCCs was measured and compared with that on Fe3O4/chitosan nanoparticles in our previous work with the same amount of Fe3O4 nanoparticles. The immobilized amount was presented by immobilized enzyme concentration which was calculated by Eq. (1): Immobilized enzyme concentration=

A1 -A2 A1

×C (1)

where A1 and A2 are the UV-absorption intensity of α-Glu before and after immobilization respectively, and C is the enzyme concentration for immobilization. As shown in Fig. 5, the amount of α-Glu immobilized on MCCs was much higher than that on Fe3O4/chitosan nanoparticles which was resulted from the high content of chitosan. 3.4 Effects of enzymatic reaction conditions on enzyme activity Both the reaction pH and temperature are of critical importance for enzymatic reaction, so these two factors were studied in order to obtain high enzyme activity. 3.4.1 Effect of reaction pH The effect of reaction pH on the relative activity of both the free and immobilized αGlu was investigated in the range of 2.0 to 8.0 at the fixed reaction temperature of 60 15

C. As shown in Fig. 6(A), both the free and immobilized α-Glu showed their

o

maximum activity at pH 4.0. The immobilized α-Glu showed higher activity over a wider pH range than the free one, indicating that the immobilization enhanced the acidic and alkaline endurances of α-Glu. The results were in accordance with the literatures [35, 36] where the immobilized enzymes also exhibited broader pH optima. This is probably due to the more stable structure and conformation of enzyme molecules caused by immobilization [35, 36]. The reaction pH was fixed at 4.0. 3.4.2 Effect of reaction temperature Fig. 6(B) shows the effect of reaction temperature on the relative activity of both the free and immobilized α-Glu in the range of 40 to 80 oC at the fixed reaction pH of 4.0. The temperature showed the same influence tendency on both the free and immobilized α-Glu. At the temperature of 60 oC, both the free and immobilized α-Glu showed the maximal catalytic activity. With the increase of temperature from 40 to 60 o

C, the enzyme activity increased. At the temperature above 60 oC, the enzyme

activity decreased as α-Glu would be inactivated at high temperature. The relative activity of the immobilized α-Glu over the free one showed that the immobilized αGlu exhibited excellent temperature endurance. This phenomenon could be attributed to the stabilization of α-Glu by the multipoint binding between α-Glu molecule and the support. Besides, the immobilized α-Glu was more rigid to retain its active structure compare with the free one [37, 38]. The temperature was set to be 60 oC for enzyme kinetic study, inhibition study and inhibitors screening.

16

3.5 Performance of the immobilized α-Glu 3.5.1 Reusability and storage stability of α-Glu The reusability of immobilized α-Glu was tested through repeated cycles. As shown in Fig. 7(A), the immobilized α-Glu retained 62.2% of its initial activity after 10 successive runs. The enzyme activity decreased with increased use and the decrease in enzyme activity could be attributed to enzyme inactivation during each cycle. Previous findings also confirmed that the immobilization enables the repeat use of enzymes. For instance, the immobilized MnP retained more than 40% of its initial activity after 7 cycles [14] and horseradish peroxidase-immobilized cross-linked enzyme aggregates (HRP-CLEAs) retained 60% of its initial activity after 7 cycles [39]. The reusability of MCCs-immobilized α-Glu was higher than that of Fe3O4/chitosan-immobilized α-Glu [28] owing to the high content of chitosan in MCCs. Results indicated that the MCCs-immobilized α-Glu performed a good reusability which would reduce the operational cost and improve the economic efficiency in practical applications. The storage stabilities of both the free and immobilized α-Glu were investigated according to Section 2.8. Fig. 7(B) shows that the immobilized α-Glu exhibited significantly greater storage stability than that of free one. The immobilized α-Glu lost only 15.7 ± 1.2% of its initial activity while the free one lost 60.5 ± 1.3% within 35 days. The results indicated that the α-Glu became more stable after multipoint binding on the support. Previously, α-Glu was immobilized on the magnetic chitosan 17

nanoparticles and the immobilized and free α-Glu lost 14.5% and 55.0% of their initial activity after 25 days [28]. The storage stability of immobilized α-Glu would guarantee their long-term preservation for further use. 3.5.2 Measurement of the kinetic parameters For enzyme kinetic parameters determination, the double reciprocal plot was constructed based on the Lineweaver-Burk plotting method [40]: 1

Km

= v v

max

1

∙ [S] + v

1 max

(2)

The linear regression equation of the double reciprocal plot (Fig. 8(A)) was 1/v = /(0.004699 ± 0.0001712) (1/[S]) + (0.005801 ± 0.0002683) (R2 = 0.9960). Each pNPG concentration was analyzed in triplicate and the average peak area of pNP was used to construct the plot. Km, the Michaelis-Menten constant, is a parameter for the evaluation of the affinity of the enzyme to the substrate. The lower the value of Km, the higher is the affinity. For immobilized α-Glu, Km was calculated to be 0.81 mM which is higher than that of the free one studied in our previous work [28]. Similar findings indicating increased Km values have been reported previously [14, 41]. The substrate diffusional hindrance caused the lower accessibility of substrate to the active site of enzyme, thus the lower affinity of enzyme to its substrate [41]. For inhibition kinetic parameter determination, acarbose was used as a model inhibitor. The Lineweaver-Burk plots for the immobilized α-Glu in the presence of acarbose at different concentrations were plotted. Seeing from Fig. 8(A), with the

18

increase of inhibitor concentration, the double reciprocal plots had unchanged Vmax and increased Km, indicating that acarbose is a competitive inhibitor. For competitive inhibitor, the Michaelis-Menten equation in double-reciprocal form was expressed as follows: 1 v

Km

=v

max

[I]

1

[1+ K ] ∙ [S] + v i

1 max

(3)

And the secondary plot equation was obtained from Eq. (3) as: Km

Slope= v

max

[I]

Km

∙K +v i

max

(4)

The secondary plot, constructed based on Eq. (4) and shown as the inset of Fig. 8(A), was used to calculate the inhibition constant Ki. The intersection of the obtained secondary plot and the x-axis is -Ki, thus, Ki was calculated to be 6.379 μM. The inhibition percentage was calculated based on Eq. (5): x

Inhibition%= (1- blank) ×100 (5) where x and blank are the peak areas of pNP measured with and without inhibitor, respectively. By varying the concentrations of acarbose at a fixed substrate concentration of 0.5 mM, the inhibition plot (Fig. 8(B)) was established. The value of IC50 was determined as 14.99 μM. The lower Ki and IC50 can be explained by the higher Km. 3.6 Inhibitors screening With the established method, enzyme inhibitors were screened from 11 TCMs and 9 vegetables with the final concentrations of 25 mg mL-1. By comparing the peak area 19

of pNP with blank control, potential inhibitors can be identified easily. The inhibition percentages for these TCMs and vegetables were separately listed in Table 1 and Table 2. In view of the results, some showed obvious inhibitory effect, such as Folium Mori in TCMs, Red Onion and Yellow Onion in vegetables. This indicated that certain inhibitors of α-Glu might exist in these extracts. The results were in accordance with those shown in the literatures, such as, flavonoids, alkaloids and polysaccharides from Folium Mori and quercetin from onion had been characterized as α-Glu inhibitors [42, 43]. These results proved that the inhibitory TCMs and vegetables could be accurately identified by the present method. 4. Conclusions MCCs were successfully used as supports for α-Glu immobilization. The immobilized α-Glu showed a broader working pH and temperature range. Improved reusability and storage stability of the immobilized α-Glu indicated that MCCs can be used as efficient supports for enzymes immobilization. Further, enzyme inhibitors were screened with the immobilized α-Glu. The results suggest that the MCCs-immobilized α-Glu is a useful tool for diverse biotechnological applications, especially in pharmaceutical and medical application fields. 5. Acknowledgements This work was funded by the National Natural Science Foundation of China (Nos. 21375136 and 21575150) and the Scholar Program of West Light Project, Chinese Academy of Sciences. 20

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Fig. 1. Schematic illustration of preparation of MCCs-immobilized α-Glu.

Fig. 2. (A) XRD pattern of Fe3O4 nanoparticles; (B) FT-IR spectra of (a) Fe3O4, (b) MCCs, (c) CS, (d) GA-modified MCCs and (e) MCCs-immobilized α-Glu.

27

Fig. 3. TEM images of (A) Fe3O4 and (B) MCCs. Inset of (B): the states of MCCs in aqueous solution without (upper) and with (lower) magnet.

Fig. 4. Effect of immobilization conditions on the relative activity of immobilized αGlu. (A) Enzyme concentration, (B) crosslinking time, (C) immobilization pH and (D) immobilization time. 28

Fig. 5. Immobilized amount of α-Glu on MCCs and Fe3O4/chitosan nanoparticles.

Fig. 6. Effect of enzymatic reaction conditions on the relative activity of immobilized and free α-Glu. (A) Enzymatic reaction pH and (B) enzymatic reaction temperature.

29

Fig. 7. (A) Reusability of immobilized α-Glu and (B) storage stability of free and immobilized α-Glu.

Fig. 8. (A) Lineweaver-Burk plots and (B) inhibition plot of immobilized α-Glu in the presence of acarbose. Inset of (A): the secondary plot for acarbose. The concentrations of acarbose: (a) 0 μM; (b) 10 μM; (c) 30 μM; (d) 50 μM.

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Table 1 Screening results of 11 TCMs against α-Glu (n=3). TCMs

% Inhibition

TCMs

% Inhibition

Barley

0

Radix et Rhizoma Rhei

52.72 ± 0.44

Cortex Moutan

10.29 ± 2.42

Radix Notoginseng

19.09 ± 2.80

Folium Mori

100

Radix Ophiopogonis

0

Phellodendron amurense

18.77 ± 0.62

Radix Puerariae

4.06 ± 1.33

Platycodon grandiflorus

20.98 ± 1.00

35.21 ± 1.02

Radix Astragali

18.81 ± 0.69

Glycyrrhiza uralensis Fisch

Table 2 Screening results of 9 vegetables against α-Glu (n=3). Vegetables

% Inhibition

Vegetables

% Inhibition

Benincasa hispida

9.74 ± 1.62

Lentinus edodes

2.05 ± 1.58

Carrot

24.09 ± 1.65

Pumpkin

3.65 ± 1.24

Chinese cabbage

15.15 ± 0.42

Red Onion

62.36 ± 0.67

Cucumber

13.55 ± 1.60

Yellow Onion

42.08 ± 1.31

Eggplant

18.16 ± 0.64

31