Synchronized purification and immobilization of his-tagged β-glucosidase via Fe3O4/PMG core/shell magnetic nanoparticles

In this paper, an efficient and convenient Fe3O4/PMG/IDA-Ni2+ nanoparticles that applied to purify and immobilize his-tagged β-glucosidase was synthesized, in which, Fe3O4/PMG (poly (N, N’-methylenebisacrylamide-co-glycidyl methacrylate) core/shell microspheres were synthesized firstly using distillation-precipitation polymerization, then iminodiacetic acid (IDA) was used to open epoxy rings on the shell of microspheres to the combination of Ni2+. The gene of β-glucosidase that was from Coptotermes formosanus Shiraki was amplified, cloned into the expression vector pET28a with an N-terminal His-tag, and expressed in E.coli BL21. The nanoparticles showed the same purification efficiency as commercial nickel column which was a frequently used method in the field of purifying his-tagged proteins from crude cell lysates. The results indicated that Fe3O4/PMG/IDA-Ni2+ nanoparticles can be considered as an excellent purification material. β-glucosidase was immobilized on the surface of Fe3O4/PMG/IDA-Ni2+ to form Fe3O4/PMG/IDA-β-glucosidase by means of covalent bound with imidazolyl and Ni2+. The immobilized β-glucosidase exhibited excellent catalytic activity and stabilities compared with free β-glucosidase. In addition, immobilized β-glucosidase can be recycled for many times and retain more than 65% of the original activity. The materials display enormous potential in the aspect of purifying and immobilizing enzyme.


Results and Discussion
Characterization of nanoparticles. According to the above-mentioned procedure, Fe 3 O 4 /PMG/IDA-Ni 2+ nanoparticles were successfully prepared. Figure 1 showed the specific procedure of nanoparticles synthesis and combination between nanoparticles and his-tagged BG. Representative TEM images of the Fe 3 O 4 and Fe 3 O 4 / PMG nanoparticles are shown in Fig. 2A,B. The Fe 3 O 4 nanoparticles have an average diameter of about 200 nm ( Fig. 2A), and were uniform both in shape and size. After being encapsulated with PMG, the size of the composite microspheres increased to about 350 nm (Fig. 2B). The obtained Fe 3 O 4 /PMG nanoparticles possessed a well-defined core shell structure and superior dispersibility in aqueous media (Fig. 2C). Figure 3 shows the FT-IR spectra of the naked and functionalized magnetic nanoparticles. As shown in Fig. 3A, the peak at 582 cm-1 was attributed to the Fe-O bond, and the peaks at about 1,618 cm −1 and 1,400 cm −1 were associated with carboxyl groups available from the stabilizer citrate. As shown in the FT-IR spectrum of the Fe 3 O 4 -MPS nanoparticles (Fig. 3B), the peak at 1,632 cm −1 corresponds to the stretching vibration of C = C bond of MPS. The successful functionalization of Fe 3 O 4 with PMG ( Fig. 3C) is demonstrated by the absorption peak of C = O at 1,721 cm −1 in GMA, and N-H at 1,528 cm −1 in MBA, respectively. These data further prove that the polymer-GMA is successfully grafted onto the Fe 3 O 4 nanoparticles surface.
Components of composite microspheres were measured by thermogravimetric analysis (TGA) (Fig. 4). While the organic components decomposited and inorganic components remained at high temperature. The 17.09 wt% loss of Fe 3 O 4 is attributed to the weight ratio of citrate stabilizer and the physically adsorbed water. After modified by MPS, the loss of 19.76 wt% was assigned to the physically adsorbed water and small amount of MPS on the magnetic surface. When the outmost PMG layer was introduced, the weight loss of the composite microspheres was about 65.07 wt%, which is much higher than Fe 3 O 4 -MPS (19.76 wt%). The first weight loss (10.3%) until 200 °C was due to the evaporation of the physically adsorbed water or solvent, and the second major weight loss (54.77%) from 200 to 600 °C was due to the decomposition of the polymer component in the shell layer of the corresponding microspheres. And the magnetite content of Fe 3 Fig. 6, respectively. The magnetic susceptibility of Fe 3 O 4 /PMG microspheres is large enough to be separated from the solution by quickly using a magnetic block. Application in protein purification. Following the above method, we tested the binding and separating ability of Fe 3 O 4 /PMG/IDA-Ni 2+ with his-tagged BG, which has a molecular weight of 56 KDa. When it comes to separating ability, a comparison was done between Ni-charged resin and Fe 3 O 4 /PMG/IDA-Ni 2+ . Figure 7A showed that nearly 97% of his-tagged BG can be purified by Ni-charged resin and Fe 3 O 4 /PMG/IDA-Ni 2+ . What is more, we can see that the purity of β -glucosidase that was gained by nanoparticles is higher than that of Ni-charged resin from lane 5 and 9. Lane 5 (Fig. 7A) had other bands except for target band that was labeled by the black arrow. However, there was only the target band in lane 9 (Fig. 7A). Further, we used the technology of western blotting to verify the purification result. In the Fig. 7B lane 5 and 9 respectively displayed an apparent band that appeared using anti his-tagged antibody. Table 1 revealed the purification fold of BG from Ni-charged resin and Fe 3 O 4 /PMG/IDA-Ni 2+ . Nickel column purification is considered to be a high-efficiency method to purify his-tagged protein. In the Table 1, purification fold of nickel column and nanoparticles was 18.3 and 17.3, respectively. The value was approximately between the two. So to some extent, we can think that purification  effect of nanoparticles is equivalent to that of nickel column. The nanoparticles will be able to be applied widely in the field of purification.
Optimal conditions of immobilization. In the assay of testing optimal conditions, the following method was used with two quantitative factors and, one variable factor. Then, a certain amount of immobilized enzyme was obtained to detect enzyme activity. The relevant graphs are shown in Fig. 8(A,B and C). The maximum amount of BG with the incubating time and incubating temperature is 120 mg BG/g carriers, 30 min and 25 °C, respectively. It was found that, with the increment of variable, relative activity gradually raised and began to reduce or kept smooth when variable got to a certain point. One reason is that the increment of amount of   enzyme leads to the reduction of combination opportunity between enzyme and nanoparticles. Each enzyme molecule competes to combine with nanoparticles, which gives rise to the partial combination between enzyme and nanoparticles within the incubation time. Another reason is that high temperature destroyed the structure of the enzyme, which causes his-tagged to be wrapped. Meanwhile, the destruction is irreversible.
At last, the immobilized BG were prepared at optimal amount of BG added (120 mg/g carriers), incubating time (30 min) and temperature (25 °C) for future study, supported by Fe 3 O 4 /PMG core/shell magnetic nanoparticles. Meanwhile, we calculated the binding capacity of Fe 3 O 4 /PMG/IDA-Ni 2+ to BG in the optimal conditions of immobilization. After incubating 30 min, the supernatant was removed. The nanoparticles loaded BG were washed using Tris-HCl buffer (50 mM, pH8.0) for some times. According to the formula in the method, we gained the binding capacity of Fe 3 O 4 /PMG/IDA-Ni 2+ to his-tagged BG is approximately 60 mg/g (BG/nanoparticles).
Effects of pH and temperature on the enzyme activity. The effects of pH and temperature on the activities of immobilized BG were studied compared to free BG. Various pH in the reaction system could affect the activity of enzymes 13 . The effects of different pH values (3.0-9.0) on the activity of free and immobilized enzymes were compared at 40 °C, and the results were displayed in Fig. 9A. The curve shows that both enzymes have the same optimal pH at pH5.5.  The effect of temperature on the catalytic rate of enzymes mainly relied on its activity. With the increment of temperature, the heat motion of the enzyme and substrate also increase. Therefore, more collisions between the substrate and the enzyme's active site occur, causing more enzyme-substrate complexes and finally more product compounds will be formed 14 . As shown in Fig. 9B, the optimal reactive temperature for free BG was similar to the immobilized BG that was 40 °C. When the temperature was higher than the optimal degree, the activities of both free and immobilized enzymes began to decrease. The immobilized BG exhibited relatively high temperature tolerance to retain 40% of its activity at 60 °C, while the free one only retained 7%. Thermal stability. In general, every kind of enzyme has its own optimum temperature. The enzymatic activity is maximal at the optimum temperature, and under or exceed the temperature enzymatic activity reduces. In consequence, finding a method to raise thermo-stability of the enzyme is imminent in order to improve their catalytic activity. After heating for 30 min, the activities of the same quality of free and immobilized enzyme were evaluated according to 2.5. As the Fig. 10B described, free BG almost lost all activity when processed temperature reached 60 °C, nevertheless, that of immobilized BG still retained 75% of its initial activity. The loss of enzyme activity could be ascribed by the change of protein structure because of heating. While the binding of Fe 3 O 4 / PMG/IDA-Ni 2+ and his-tagged BG avoid heat damage to some extent, accordingly active site of the enzyme is protected, which brought down the harm of high temperature. The similar phenomena had been discovered. For example, Gupta et al. prepared Cu-IDA and Cu-IDA-Sepharose that was used to immobilize bromelain 15 . Yang et al. applied Ni 2+ -PD-MNPs to immobilize his-tagged red fluorescent protein 16 . In their experiments, immobilized enzyme showed excellent thermal stability at high temperature (from 50 to 80 °C) compared to the free enzyme.  Storage stability. Two copies of free and immobilized BG were prepared in advance. One copy of that stored in 4 °C, and another copy stored in 25 °C. The equal quality of both enzymes was used for the detection of enzymatic activity every four days until to 20th days. The results were depicted in Fig. 10C.
The storage stability of the immobilized BG was apparently higher than that of the free. Both BGs storing in 4 °C maintained the higher activities than those in 25 °C. The immobilized BG stored at 4 °C kept 91% of its original activity, and the free enzyme retained 80% of its activity at 4 °C. Free BG lost 80% of its primal activity at 25 °C at 20 days, the immobilized BG lost only about 25% of its activity. It suggests that the magnetism of Fe 3 O 4 /PMG/ IDA-Ni 2+ nanoparticles could hold BG in a stable state in comparison to the free enzyme.
Reusability assay. Reusability of immobilized enzymes is a significant parameter when it comes to the significance of immobilization. Reusability of immobilized BG in this research was evidenced by its surplus activities at each round iteration. In every round, immobilized BG was incubated with the 4 mM p-NPG for 10 min, and then the reaction was ceased using 1 M sodium carbonate. Fe 3 O 4 /PMG/IDA-BG was separated by an external magnet and washed several times with Tris-HCl buffer (50 mM, pH8.0). Immediately following, sedimentary immobilized BG was suspended by Tris-HCl buffer (50 mM, pH8.0) and entered into the next round of usage. The cycle batch of immobilized BG is eleven times, and its activity retained approximately 70% as illustrated in Fig. 11. The excellent reusability could significantly reduce the operation cost in practical applications 17 . It could be explained by that immobilization of BG limited its freedom to resist conformational changes, and hence led to increasing stability toward denaturation.
In consideration of some excellent properties of immobilized BG in this research, a comparison was carried out between our immobilized enzyme and other magnetic immobilized enzymes that were reported in literature. There mainly included four aspects: magnetic saturation (Ms), binding capacity, thermal stability and retained activity of the reusage. The corresponding data was showed in the Table 2. These magnetic nanoparticles exhibited different advantages. Our magnetic nanoparticles are prior to others in the aspects of binding capacity and reusability by the comparison.
Kinetic parameters. p-NPG was used as substrate in the BG activity assays. Kinetic parameters, the Michaelis constant (K m ), the maximum rate of the reaction (V max ) and the catalytic constant (K cat ) for free and immobilized BG were measured using p-NPG at 2-16 mM concentrations. K m and V max were calculated from the  Line weaver-Burk plots using the initial rate of the reaction data. From Table 3, we gained the affinity of immobilized BG to substrates is higher than that of free one, but for catalytic activity, the result was just the opposite.
where [S] is the concentration of the substrate, v and V max represent the initial and the maximal rate of the reaction, respectively. K m is defined as the substrate concentration when reaction speed is equal to one half of the maximum reaction rate. K m can reflect the affinity of the enzyme and substrate. The lower K m is, the greater the affinity is. K cat stands for catalytic constant. The bigger K cat is the catalytic activity for substrates is better.

Conclusion
In summary, a magnetic core-shell nanostructure, in which Fe 3 O 4 magnetite is the core and PMG is the shell layer, has been synthesized by distillation precipitation polymerization. Fe 3 O 4 /PMG/IDA-Ni 2+ nanoparticles exhibited better performance in the separation of His-tagged BG than that of nickel column purification. Especially, the binding capacity can reach to 60 mg/g. Compared with free BG, the immobilized BG showed stronger temperature resistances, better repeatability and stability. It indicates that the immobilized BG on the Fe 3 O 4 /PMG/ IDA-Ni 2+ nanoparticles have a potential application in the field of catalyst, such as improving flavor juice, degrading cellulose and defensing pest.

Methods
Materials. Iron

Construction of Recombinant Expression Plasmids and Expression of His-tagged BG.
The coding sequences of BG (no. GQ911585) genes were searched from NCBI-GenBank and used to design primers (Table 4) 1 . The signal peptide and restriction enzyme cutting sites were analyzed by SignalP 3.0 Server and Webcutter 2.0, respectively. cDNA of Coptotermes formosanus Shitake was served as template to amplify the gene of BG by PCR technology. Following this, the PCR product was digested using two kinds of restriction endonuclease-Hind Ш and Xho Ι . Finally, the digested product was cloned to expression vector pET28a between the Hind Ш and Xho Ι restriction sites. The recombinant plasmid was verified by DNA sequencing. The empty plasmid (only pET28a) and a recombinant plasmid expressing BG were transformed into BL21 (DE3). A signal colony was incubated in LB media with kanamycin by shaking at 37 °C overnight. Enlarged culture was done according to 1% between bacteria and LB media by shaking at 37 °C until OD 600 = 0.4, at which time the temperature of shaker was lowered to 25 °C, isopropyl-β -D-thiogalatopyranoside (IPTG) was added to a final concentration of 0.2 mM. After shaking about 6 h, the cells were harvested by centrifugation at 4,500 g and 4 °C and stored at − 80 °C.
The frozen cell was resuspended in Tris-HCl (50 mM, pH8.0) after being washed using Tris-HCl (50 mM, pH8.0) for twice, and then treated 30 min with lysozyme whose final concentration is 1 mg/mL on ice. At last, the suspension was broken by Ultrasonic Cell Disruptor at 30 min, in which precipitation and supernatant were separated by centrifugation at 12,000 rpm and 4 °C for 20 min twice and stored at − 80 °C.

Loading amount of Fe 3 O 4 /PMG/IDA to his-tagged BG.
A certain volume of the protein solution was mixed with nanoparticles and incubated at 25 °C for 30 min. Subsequently, the supernatant was removed, and nanoparticles binding protein were washed with Tris-HCl buffer (50 mM, pH8.0) for some times until the concentration of the supernatant is zero. The amount of immobilized BG was determined by subtracting the amount of BG remaining in the Tris-HCl buffer from the BG added to immobilization. The BG loading amount was calculated from the following equation: x x 0 0 1 1  where C 0,1…x is the protein concentration and V 0,1…x is the volume of the free BG solution added to immobilization, respectively. m is the amount of nanoparticles.
Western blotting. Protein samples from separation of nickel column and nanoparticles were separated by SDS-PAGE and transferred to PVDF membrane. The membrane was subsequently blocked and incubated with primary antibody at 4 °C overnight. Anti-His-tag (1:2,000) was purchased from Abmart (Shanghai, China). The secondary antibody, anti-mouse (1:5000) was from Zhongshan Golden Bridge Biotechnology (Beijing, China). The blots were exposed to ECL Western Blotting Substrate (Vazyme, Nanjing, China).

BG immobilization.
Initially, three main factors were considered, including the amount of BG added, tem- Assay of β-glucosidase activity. The enzymatic assays of p-NPG were performed as follows: the certain amounts of free and immobilized BG were incubated 10 min with 4 mM p-NPG substrates, 50 mM NaAc-HAc buffer at 40 °C. Assays were stopped by the addition of 1 M sodium carbonate. The measurement of color change was performed at 410 nm using spectrophotometer. The enzymatic assays were done three times, respectively. The final concentration of p-NP and corresponding value of OD 410 were used for the standard curve construction 18 .
The specific activities (U/mg) of both free and immobilized BG were calculated with the following formula: where V reaction is the total volume of reaction; t is the reaction time; m is the mass of free and immobilized BG. The enzymatic activity assays results were calculated in accordance with 1 mg enzyme and the quantity of product per unit time.