Magnetic combined cross-linked enzyme aggregates of horseradish peroxidase and glucose oxidase: an efficient biocatalyst for dye decolourization

Abstract

In this study, the magnetic combined cross-linked enzyme aggregates (combi-CLEAs) of glucose oxidase (GOD) and horseradish peroxidase (HRP) were designed and prepared successfully. The magnetic combi-CLEAs were fabricated by co-precipitation and cross-linking for the immobilization of GOD and HRP onto amino-functionalized Fe₃O₄ particles. Ethanol and glutaraldehyde were used as precipitant and cross-linker, respectively. In order to evaluate the catalytic performance, the magnetic combi-CLEAs were applied in the removal of direct black-38 (DB38). The results indicated that the magnetic combi-CLEAs exhibited high removal efficiency for DB38 at pH 6.0 and 40 °C. Under optimal conditions, the highest removal efficiency of DB38 was 92.28%, which was higher than that of free enzymes (46.82%). After 8 cycles of reuse, the DB38 removal efficiency could still reach 55.53%, indicating that the magnetic combi-CLEAs had good reusability. Due to the easy separation and high reusability, this study shows that magnetic combi-CLEAs may potentially be an environmentally friendly and cost effective biocatalyst for dye removal and water treatments.

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Introduction

Owing to the advantages of cross-linked enzyme aggregates (CLEAs), such as improved operational and thermal stability, highly concentrated enzyme activity, as well as the property of repeated utilization,^1,2 more and more attention has been paid to this cost-effective immobilization approach. Recently, co-immobilization of two or more enzymes in single CLEAs for the sole purpose of performing two or more biotransformations in sequence has been developed to improve the synergistic interactions between multi-enzymes. This form of CLEAs has been named combi-CLEAs. In combi-CLEAs, different enzymes can be simultaneously confined in the same aggregate and perform a versatile cascade biotransformation/bioconversion. These combi-CLEAs have several advantages including simple preparation process, high catalytic performance, increased space-time yields, as well as low production cost. Until now, many kinds of combi-CLEAs have been developed for cascade catalysis, such as combi-CLEAs of ketoreductase and D-glucose dehydrogenase for cofactor regeneration in the synthesis of chiral alcohols,³ combi-CLEAs of glucoamylase and pullulanase as catalyst for starch saccharification,⁴ combi-CLEAs of alpha-amylase, glucoamylase and pullulanase used for one pot starch hydrolysis,⁵ combi-CLEAs of xylanase and mannanase applied for hydrolysis of lime-preteated sugarcane bagasse and milled corn stover,⁶ etc. However, the separation of combi-CLEAs from reaction mixtures (which are always in the form of suspension) is not feasible using conventional centrifugation or filtration techniques, which always result in the increased size of CLEAs clusters or CLEAs clumping.^7–9 In order to decrease clumping of CLEAs during separation, magnetite nanoparticles have been used to prepare magnetic CLEAs,^7,10 which can be easily separated from the reaction mixture by using magnetic fields, thereby eliminating the need of filters and centrifugal techniques.

The enzymes with low superficial Lys residues cannot be properly cross-linked and further result in the obtained CLEAs with low mechanical stability or even releasing enzyme into the reaction media during the reaction process. In order to overcome the drawbacks about low Lys residue contents of enzyme, some polymers containing numerous free amino groups or proteins rich in Lys residues such as poly-lysine,¹¹ polyethyleneimine,¹² bovine serum albumin (BSA)¹³ and hen egg white¹⁴ are added during the preparation of CLEAs. Thus, the preparation of magnetic combi-CLEAs with additives not only improves the cross-linking efficiency and mechanical stability, but also offers the possibility to realize the facile and easier magnetic separation of the biocatalyst.⁷

Dyes, which are widely used in textiles, paper, rubber and plastics industries, impart colors to water sources and then create environmental pollutions that severely influence the quality of life and living organisms.¹⁵ Thus, developing efficient technologies for the removal of dyes from wastewater are urgent. Enzymatic degradation and decolorization of dyes is an environment friendly and cost competitive alternative to chemical decomposition processes. For example, horseradish peroxidase (HRP) is suitable for dyes degradation and decolourisation because of its high catalytic activity, commercial availability, broad spectrum of substrates, and low cost with respect to other enzymes. The HRP-catalyzed oxidation of dyes needs hydrogen peroxide (H₂O₂) as the oxidizing agent. The continuous addition of H₂O₂ will cause the inactivation of HRP owing to high local concentrations. This drawback can be avoided by in situ formation of H₂O₂ from glucose catalyzed by glucose oxidase (GOD).

Thus, in the present work, the preparation of the magnetic GOD and HRP combi-CLEAs and its application in dye removal were studied. The magnetic combi-CLEAs were prepared by co-precipitation and cross-linking of GOD and HRP onto amino-functionalized Fe₃O₄ particles. The effects of precipitant agents, cross-linking time and cross-linker concentration on the activity recovery were investigated. The magnetic combi-CLEAs were used in direct black-38 (DB38) removal reactions. The reaction conditions including the activity ratio of GOD to HRP, substrate concentration, pH and temperature were investigated detailedly. Additionally, the reusability of the magnetic combi-CLEAs was also studied. Compared to the single enzymatic process, some advantages were obtained when this combi-CLEAs system was used: (1) the first enzyme (GOD) generated H₂O₂ by catalyzing the oxidation of glucose, while the second one (HRP) used H₂O₂ (as a substrate) in the degradation of dye; (2) the in situ formation of H₂O₂ could reduce the peroxide-induced inactivation of HRP.

Material and methods

Materials

GOD (E.C.1.1.3.4, from Aspergillus niger), HRP (E.C.1.11.1.7), BSA and DB38 were purchased from Sigma-Aldrich (America). 3-Amino-propyl triethoxysilane (APTES) was purchased from Beijing Shenda Fine Chemical Co., Ltd (China). Ammonium sulfate, ethanol, isopropanol, n-butyl alcohol, acetone and 1,2-dimethoxyethane (DME) were purchased from Dingguo Biotech Co. (China). All other chemicals were of analytical grade and used without any further purification.

Preparation of amino-functionalized Fe₃O₄ particles

The Fe₃O₄ particles were prepared according to the previous literature.¹⁶ Briefly, FeCl₃·6H₂O (0.04 mol) and FeCl₂·4H₂O (0.02 mol) were dissolved in 50 mL of HCl solution (0.5 M), followed by the drop-wise addition of ammonium hydroxide (0.1 M) under agitation at 25 °C until visible precipitates were obtained. The resulting precipitates were washed several times with deionized water, and then dried at 70 °C for 24 h. After that, the obtained Fe₃O₄ particles were functionalized with APTES by a silanization reaction.¹⁷ Briefly, the Fe₃O₄ particles (0.50 g), APTES (2.50 mL) and deionized water (0.63 mL) were added into anhydrous ethyl alcohol (62.5 mL), and the mixture was sonicated for 30 min. After that, glycerol (37.5 mL) was added. The solution was incubated under vigorously stirring for 6 h at 90 °C. The obtained precipitate (defined as amino-functionalized Fe₃O₄ particles) was separated by a permanent magnet, washed with copious ethanol and water successively and then dried at 50 °C for 24 h.

Preparation of magnetic combi-CLEAs

Magnetic combi-CLEAs of GOD and HRP were prepared by co-precipitation method. Typically, 1 mL of BSA solution (10 mg mL⁻¹) and 5 mg of amino-functionalized Fe₃O₄ particles were mixed with 1 mL of phosphate buffer solution (PBS, 0.1 M) containing GOD and HRP (3 mg mL⁻¹ of HRP, the activity ratio of GOD : HRP = 1 : 1–3 : 1 (U/U)), and then nine-fold volume of precipitants (chilled organic solvents or saturated ammonium sulfate solution) were added into the above mixture with stirring for 30 min at 4 °C. After that, 1 mL of chilled glutaraldehyde was added under stirring and cross-linked for time was 1–5 h at 4 °C. Finally, the magnetic combi-CLEAs were separated from the mixture, washed three times with PBS and stored at 4 °C.

The effects of precipitation agents (ammonium sulfate, ethanol, iso-propanol, n-butanol, acetone and DME), concentration of glutaraldehyde, and cross-linking time on the activity recovery were investigated.

Activity assay

The activities of free GOD or GOD-CLEAs were performed by monitoring the oxidation of glucose according to the colorimetric method reported in the previous literatures.^18,19 Briefly, 1.5 mL of 13% glucose and 1.5 mL of PBS (pH 7.0, 0.2 M) containing HRP (0.25 mg), 4-aminoantipyrine (0.25 mg) and phenol (0.003 mg) were added to the GOD sample at 25 °C. The reaction mixture was vigorously shaken for 30 s at 25 °C. The molar concentration of the product was measured using a UV-Vis spectrophotometer (Thermo 300) at 500 nm.

The activities of free HRP or HRP-CLEAs were measured by monitoring the conversion of H₂O₂ using the method of Worthington with a slight modification. Typically, 1.5 mL of H₂O₂ solution (1.7 mM, prepared freshly) and 1.4 mL of PBS containing 4-aminoantipyrine (2.5 mM) and phenol (0.17 M) were mixed with the HRP samples at 25 °C under agitation. The reaction mixture was allowed to stand for 30 s at 25 °C under vigorous shaking condition, and the molar concentration of the product was measured at 500 nm.

One unit (U) of enzyme activity was defined as the amount of enzyme required to catalyze the conversion of substrate (1 μmol, glucose or H₂O₂) to end-product per min at 25 °C. The activity recovery of CLEAs was calculated as the ratio of the specific activity of CLEAs (or residual activity) to the initial activity of free enzyme.

Characterization of magnetic combi-CLEAs

Scanning electron microscopy (SEM, Nova Nano SEM 450) was used to characterize the morphology of magnetic combi-CLEAs.

Application of magnetic combi-CLEAs in DB38 removal

The potential for the magnetic combi-CLEAs in treating dye was evaluated by its application in DB38 removal. The enzymatic removal of DB38 was carried out by mixing 1 mL of glucose (0.25–2%), 1 mL of DB38 (80 mg mL⁻¹), and the magnetic combi-CLEAs at stirring speeds of 200 rpm at 25–55 °C and pH 4.0–8.0. The magnetic combi-CLEAs were isolated using a permanent magnetic field after 9 h reaction and washed twice with PBS. The supernatant was collected, and then the residual concentration of DB38 was measured at 550 nm. Control tests were run in each experiment under the same experimental conditions. All assays were performed in triplicate. The removal efficiency was calculated by measuring the content of DB38 in the original solution, supernatant and collected washing solutions.

To determine the optimal conditions for DB38 removal, the activity ratio of GOD to HRP, substrate concentration, pH and temperature were optimized.

The reusability of the magnetic combi-CLEAs was measured through repeated uses in the removal reaction of DB38. After each reaction cycle, the magnetic combi-CLEAs were recovered with a magnet, and washed with PBS and re-introduced into the fresh reaction medium for another assay run.

Results and discussion

Preparation and characterization of magnetic combi-CLEAs

Effect of precipitant on activity recovery of magnetic GOD-CLEAs and HRP-CLEAs. To evaluate the ability of precipitants, six precipitants were added to the enzyme solution to obtain physical aggregates of enzyme. After that, the physical aggregates were redissolved, and then the activity recovery was measured. Results in Table 1 indicated that the activity recovery of the redissolved HRP and GOD aggregates using some organic solvents (iso-propanol, n-butanol, acetone and DME) was low which might be caused by partial denaturation of the enzymes. The best results were obtained using saturated ammonium sulfate solution and ethanol as precipitants, giving an activity recovery of 133.31% and 106.83% for the redissolved HRP and GOD aggregates, respectively. But, when saturated ammonium sulfate solution was used as precipitant, the activity recovery for the redissolved GOD aggregates was only 39.88%. However, when ethanol was used as precipitant, the highest activity recovery for the redissolved GOD aggregates was obtained, and the activity recovery of the redissolved HRP aggregates (127.51%) was only slightly lower than that of saturated ammonium sulfate solution as precipitant. Additionally, the results also showed that the activities of redissolved GOD and HRP aggregates were higher than the initial one, which can be explained by that the natural inhibitors or additives presented in the original enzyme sample were removed during precipitation process.²⁰ Similar phenomena were also observed for laccases, lipases, trypsin and peroxidase in the previous reports.^20–22

Table 1 Effect of precipitant on activity recoveries of redissolved GOD and HRP^a

Precipitant type	Activity recovery of redissolved GOD aggregates (%)	Activity recovery of redissolved HRP aggregates (%)
a The preparation parameters: the ratio of enzyme solution to precipitant with 1 : 9 (v/v), and enzyme precipitated for 30 min at 4 °C.
Saturated ammonium sulfate	39.88 ± 1.05	133.31 ± 2.72
Ethanol	106.83 ± 2.36	127.51 ± 2.69
Iso-propanol	91.60 ± 2.17	78.44 ± 1.92
n-Butanol	78.51 ± 1.96	98.07 ± 2.31
Acetone	56.63 ± 1.61	59.70 ± 1.70
DME	41.94 ± 1.11	33.06 ± 0.88

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The activity recoveries of magnetic GOD-CLEAs and HRP-CLEAs were further measured. As shown in Table 2, the results indicated that the magnetic GOD-CLEAs showed relative high activity recovery (18.53%) with ethanol as precipitant. In addition, the activity recovery of the magnetic HRP-CLEAs (24.05%) was only slightly lower than that of saturated ammonium sulfate solution as precipitant, but higher than that of iso-propanol, n-butanol, acetone and DME as precipitants. Therefore, ethanol was chosen as a suitable precipitant for magnetic CLEAs preparation in subsequent experiments.

Table 2 Effect of precipitants on activity recoveries of magnetic GOD-CLEAs and HRP-CLEAs^a

Precipitant type	Activity recovery of magnetic GOD-CLEAs (%)	Activity recovery of magnetic HRP-CLEAs (%)
a The preparation parameters: the ratio of enzyme solution to precipitant with 1 : 9 (v/v), and enzyme precipitated for 30 min at 4 °C and cross-linked using glutaraldehyde (60 mM) as cross-linker for 3 h at 4 °C.
Saturated ammonium sulfate	8.06 ± 0.16	25.17 ± 0.49
Ethanol	18.53 ± 0.37	24.05 ± 0.48
Iso-propanol	16.55 ± 0.33	17.76 ± 0.35
n-Butanol	15.59 ± 0.31	20.56 ± 0.41
Acetone	10.83 ± 0.22	9.31 ± 0.18
DME	7.61 ± 0.15	5.30 ± 0.11

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Effect of cross-linking time and cross-linker concentration on activity recovery of magnetic GOD-CLEAs and HRP-CLEAs. The selection of the cross-linking time and cross-linker concentration plays a critical role in the preparation of CLEAs with high cross-linking efficiency. Glutaraldehyde is one of the most widely used cross-linker in the preparation of CLEAs. To achieve effective cross-linking between the precipitated enzyme and amino group of the functionalized Fe₃O₄ particles, the effects of cross-linking time and glutaraldehyde concentration on the activity of the resultant CLEAs were studied.

The activity recovery of the enzyme was assayed after subjecting the enzyme to cross-linking in the range from 1 to 9 h at 4 °C, and the results were shown in Table 3. When the cross-linking time was 1 h, the activity recoveries of the magnetic GOD-CLEAs and HRP-CLEAs were 13.98% and 9.55%, respectively. The low activity recovery could be ascribed to the insufficient cross-linking of the enzyme molecules and amino-functionalized Fe₃O₄ particles, which led to unstable aggregation and leakage of enzyme.²³ With the increase of cross-linking time up to 4 h, the activity recoveries of GOD-CLEAs and HRP-CLEAs were significantly increased. When cross-linking time exceed 4 h, the activity recoveries of the magnetic GOD-CLEAs and HRP-CLEAs started to decrease, which suggested that longer cross-linking times did not lead to a further increase in activity recovery but to a slight decrease due to excessive cross-linking of between the precipitated enzyme and amino group of the functionalized Fe₃O₄ particles and excessive exposure to glutaraldehyde. The excessive cross-linking restrained the enzyme's flexibility, limited mass transfer, and thus resulted in reduction of enzymatic activity.²⁰ The excessive exposure to glutaraldehyde might also result in denaturation of the enzyme.⁶ Thus, when cross-linking time was 4 h, the maximum activity recoveries of 20.04% and 27.66% for the magnetic GOD-CLEAs and HRP-CLEAs were obtained.

Table 3 Effect of cross-linking time on activity recoveries of magnetic HRP-CLEAs and GOD-CLEAs^a

Cross-linking time (h)	Activity recovery of magnetic GOD-CLEAs (%)	Activity recovery of magnetic HRP-CLEAs (%)
a The preparation parameters: the ratio of enzyme solution to precipitant with 1 : 9 (v/v), and enzyme precipitated using ethanol as precipitants for 30 min at 4 °C and cross-linked at 4 °C using glutaraldehyde (60 mM) as cross-linker.
1	13.98 ± 0.27	9.55 ± 0.19
2	17.39 ± 0.34	15.92 ± 0.31
3	18.53 ± 0.37	24.05 ± 0.48
4	20.04 ± 0.40	27.66 ± 0.55
5	19.14 ± 0.38	26.22 ± 0.52

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The concentration of glutaraldehyde could influence the performance of CLEAs, therefore, different concentrations of glutaraldehyde (30–150 mM) were employed to prepare CLEAs. The results were shown in Table 4. With increase of glutaraldehyde concentration, the activity recoveries of the magnetic GOD-CLEAs and HRP-CLEAs increased firstly and then decreased. When the glutaraldehyde concentration was 90 mM, the highest activity recoveries for the magnetic GOD-CLEAs (22.47%) and HRP-CLEAs (30.51%) were achieved. When the glutaraldehyde concentrations were lower than 90 mM, the crosslinking between the enzyme molecules and amino-functionalized Fe₃O₄ particles may be insufficient, and the enzyme molecule may be too flexible which resulted in unstable aggregation and low activity.²⁴ When the glutaraldehyde concentrations were above 90 mM, extremely cross-linking may be occurred which led to a higher size of the magnetic GOD-CLEAs and HRP-CLEAs, reduced their surface area, and then reduced activity recovery.⁵ On the other hand, glutaraldehyde as a small molecule could penetrate into the internal structure of the enzyme and react with the amino acid residues of the catalytic site, which resulted in an unfavorable cross-linking of catalytic-important amino acid residues, and then inactivated the enzyme.²⁵ Therefore, 90 mM of glutaraldehyde solution was chosen as the optimal concentration for the preparation of magnetic GOD-CLEAs and HRP-CLEAs in the subsequent experiments.

Table 4 Effect of glutaraldehyde concentration on activity recoveries of magnetic GOD-CLEAs and HRP-CLEAs^a

Glutaraldehyde concentration (mM)	Activity recovery of magnetic GOD-CLEAs (%)	Activity recovery of magnetic HRP-CLEAs (%)
a Experiments were performed using ethanol as precipitants for 30 min at 4 °C and glutaraldehyde as cross-linker for 4 h at 4 °C, respectively.
30	7.74 ± 0.15	15.74 ± 0.31
60	20.04 ± 0.40	27.66 ± 0.55
90	22.47 ± 0.44	30.51 ± 0.61
120	21.78 ± 0.43	28.81 ± 0.57
150	20.54 ± 0.41	27.52 ± 0.55

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Effect of preparation conditions on activity recovery of GOD and HRP in magnetic combi-CLEAs. On the basis of these optimum preparation conditions mentioned above, the activity recovery of co-immobilized GOD and HRP was determined. The results indicated that 21.32% and 29.83% of activity recovery were achieved for GOD and HRP respectively in the magnetic combi-CLEAs preparation, which was similar to that of the magnetic GOD-CLEAs (22.47%) and HRP-CLEAs (30.51%). The results indicated that there was no significant difference of preparation conditions between the magnetic combi-CLEAs and the magnetic CLEAs. Therefore, optimum preparation conditions including chilled ethanol as precipitating agent, final glutaraldehyde concentration of 90 mM and cross-linking time of 4 h were chosen for the subsequent magnetic combi-CLEAs preparation.

Characterization of magnetic combi-CLEAs

The magnetic combi-CLEAs were separated by a permanent magnet, and the photos were shown in Fig. S1.† Due to inclusion of magnetite nanoparticles into combi-CLEAs, the resultant combi-CLEAs became magnetic which facilitated the separation from the mixture using magnet. The morphology of the magnetic combi-CLEAs was investigated by SEM. CLEAs had usually two morphologies: spherical appearance and less-structured form.⁷ Fig. 1 indicated that the magnetic combi-CLEAs due to the inclusion of magnetite nanoparticles were mostly spherical particles with an average diameter of 31 ± 6 nm.

Fig. 1 SEM of magnetic combi-CLEAs.

Removal of DB38 catalyzed by magnetic combi-CLEAs

Effect of activity ratio of GOD to HRP on DB38 removal efficiency. Catalysis cascade reactions have several advantages such as drastically decreasing consumption of auxiliary chemicals, reducing operating time and costs, etc. In this work, GOD was used to generate H₂O₂, and then, H₂O₂ served as a substrate for HRP to remove DB38. The effect of the activity ratio of GOD to HRP in the magnetic combi-CLEAs on DB38 removal efficiency was studied (Fig. 2). At lower activity ratio of GOD to HRP, the removal efficiency of DB38 was limited due to the available H₂O₂. With an increase of the activity ratio of GOD to HRP, the removal efficiency of the DB38 increased. When the activity ratio was 2.5 : 1, 71.21% of the removal efficiency of the DB38 was obtained after 9 h reaction. Further increase the activity ratio up to 3 : 1 didn't result in a significant increase of removal efficiency. Therefore, the optimization of the activity ratio (2.5 : 1) was used in the subsequent experiments.

Fig. 2 The effect of the activity ratio of GOD to HRP on the DB38 removal efficiency. The reactions were performed at 25 °C. The concentrations of HRP and glucose were 3 mg mL⁻¹ and 2% at pH 7.0, respectively.

Effect of glucose concentration on DB38 removal efficiency. Glucose concentration as an essential parameter had great influence on the DB38 removal efficiency. To analyze the effect of glucose concentration on the DB38 removal efficiency, the experiments were carried out by varying glucose concentration from 0.25% to 2%. Fig. 3 demonstrated that increasing the glucose concentration resulted in the improvement of the DB38 removal efficiency. The reason was due to the increase of the glucose concentration resulting in higher H₂O₂ concentration which could be attributed to improve reaction rate of the DB38 degradation.⁵ When the glucose concentration was increased to 1%, the maximum removal efficiency was achieved. Further increasing the glucose concentration didn't result in the further improvement of the removal efficiency due to the limited availability of the magnetic combi-CLEAs for the DB38. Thus, the subsequent experiments were carried out with a fixed glucose concentration (1%).

Fig. 3 The effect of glucose concentration on the DB38 removal efficiency. The reactions were performed at 25 °C. The concentrations of HRP and activity ratio of GOD to HRP were 3 mg mL⁻¹ and 2.5 : 1 at pH 7.0, respectively.

Effect of temperature on DB38 removal efficiency. The effect of temperature on DB38 removal efficiency was illustrated in Fig. 4. At 25–55 °C, the following effects of temperature profiles were observed. The DB38 removal efficiency at 25 °C was only 70.50%, which could be ascribed to that the low temperature affected the combination of enzyme and substrate and dissociation of some radicals in enzyme,²⁶ and then inhibited the DB38 removal.²⁷ An increase in temperature increased the DB38 removal efficiency, which can be attributed to the reaction rate increased thermodynamically with the increase of the temperature. The maximum removal efficiency (86.22%) was achieved at 40 °C. When the temperature was beyond 40 °C, the removal efficiency decreased gradually. As the temperature was increased to 55 °C, the DB38 removal efficiency was decreased to about 74.37%. This decrease could be explained as follows: when the temperature was beyond the optimum temperature, the conformation changes of the enzyme at the high temperatures would reduce the enzymatic activity and hence the reaction rate. Additionally, the increase of the temperature lowered the concentration of dissolved oxygen and increased the self-decomposition of the generated H₂O₂.²⁸ Therefore, the subsequent experiments were carried out at 40 °C.

Fig. 4 The effect of temperature on DB38 removal efficiency. The concentrations of HRP and glucose were 3 mg mL⁻¹ and 1% at pH 7.0, respectively. The activity ratio of GOD to HRP was 2.5 : 1.

Effect of pH on DB38 removal efficiency. The DB38 removal efficiency mostly depended on enzymatic activity, and most enzymes had a characteristic pH value at which their activity was maximized. The interrelation of enzyme activity with pH relied on the acidic–basic behavior of the reaction system. To study the effect of pH on the removal efficiency of the DB38, the reaction solutions with various pH values ranging from 4.0 to 8.0 were studied. As shown in Fig. 5, the magnetic combi-CLEAs were highly efficient in the wide pH range of 4.0–7.0, which was wider than that reported by Zoran Vujčić and coworkers.²⁹ They studied the enzymatic decolorization of the dyes at different pH values, and concluded that majority of dyes were successfully decolorized in the pH range of 5.0–7.0 for HRP. The reason for the wider pH range laid in the fact that the simultaneous usage and immobilization of GOD and HRP widened the optimal pH range. Fig. 5 also showed the optimum pH for DB38 removal was 6.0, and the maximum removal efficiency was 92.28%. When pH was increased to 8.0, the removal efficiency was decreased greatly. According to the previous studies, at lower pH (pH < 4.0), the removal efficiency was low which was because HRP was less stable due to dissociation of heme group from its pocket.^30,31 Increasing pH value to 8.0 could also lead to the rapid decrease of the removal efficiency because the GOD and HRP activity was inhibited or enzymes denatured under basic conditions.^32,33

Fig. 5 The effect of pH on DB38 removal efficiency. The reactions were performed at 40 °C. The concentrations of HRP and glucose were 3 mg mL⁻¹ and 1%, respectively. The activity ratio of GOD to HRP was 2.5 : 1.

Reusability of magnetic combi-CLEAs. CLEAs offer a unique opportunity for the recycling and reuse of enzymes through their coupling to magnetite particles. The reusability of the magnetic combi-CLEAs could improve DB38 removal efficiency and made the DB38 removal process more efficient and cost-effective. Fig. 6 showed the reusability of the magnetic combi-CLEAs. At the first cycle of reaction, the DB38 removal efficiency was 92.28%; after 8 cycles, the removal efficiency still remained 55.53%. While for the magnetic HRP-CLEAs, the removal efficiency for DB38 was 82.76% for the first time under the same conditions, and was 50.81% after 8 consecutive operations. The observed decrease of removal efficiency could be due to mechanical losses and denaturation of CLEAs during the washing procedure.³⁴ But, compared with the magnetic HRP-CLEAs, the magnetic combi-CLEAs had better removal efficiency for DB38. At the same time, the DB38 removal by the free enzyme was also measured. The removal efficiency was only 46.82% for the first time. The observed high reusability of the magnetic combi-CLEAs was partially due to the easy and almost full recovery of the biocatalyst.⁷ Other reason was the strong attachment of bienzymes on the Fe₃O₄ particles,³⁵ giving no detectable enzyme leakage during reactions and recycling experiments. Furthermore, in order to measure the effect of the amino-functionalized Fe₃O₄ (Fe₃O₄–NH₂) particles for the DB38 removal, the removal efficiency was measured under the same conditions, and 13.15% of removal efficiency was observed for the first cycle which was attributed to the adsorption of DB38 on the amino-functionalized Fe₃O₄ particles. Compared to its adsorption, the degradation of the DB38 catalyzed by the magnetic combi-CLEAs played a dominant role.

Fig. 6 Reusability of enzyme and Fe₃O₄–NH₂ on DB38 removal. The reactions were performed at 40 °C. The concentrations of HRP and glucose were 3 mg mL⁻¹ and 1% at pH 6.0, respectively. The activity ratio of GOD to HRP was 2.5 : 1.

Conclusions

In this study, the magnetic combi-CLEAs were prepared by co-precipitation and cross-linking of GOD, HRP and amino-functionalized Fe₃O₄ particles. The effect of the precipitant type, concentration of glutaraldehyde and cross-linking time on the activity recovery of the magnetic combi-CLEAs were investigated. The magnetic combi-CLEAs were used to remove DB38, and the results indicated that the combi-CLEAs exhibited high removal efficiency and good reusability. Thus, the present study provides a stable catalyst for dye removal. Given the wide variety of possible enzyme combinations and the efficiency of this combi-CLEAs preparation method, we believe that this work presents a new approach to the synthesis of multienzyme biocatalysts for catalysis of multi-step cascade reactions.

Acknowledgements

This work was supported by the National Nature Science Foundation of China (No. 21306039, 21276060, 21276062), the Natural Science Foundation of Hebei Province (B2015202082, B2016202027) and Tianjin City High School Science & Technology Fund Planning Project (20140513).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra12009a

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