Immobilization of Agaricus Bisporus Laccase on Ceramic-Chitosan Composite Support and Their Properties: Potential for Oily Wastewater Treatment

Li Zhilin; Shang Weilong; Liu Wei; Li Hesheng

(1. College of Materials Science and Engineering, Beijing University of Chemical Technology, Beijing 100029; 2. Beijing Key Laboratory of Membrane Science and Technology, College of Chemical Engineering, Beijing University of Chemical Technology, Beijing 100029; 3. Research Institute of Yanshan Petrochemical Co., SINOPEC, Beijing 102500)

Immobilization of Agaricus Bisporus Laccase on Ceramic-Chitosan Composite Support and Their Properties: Potential for Oily Wastewater Treatment

Li Zhilin1; Shang Weilong2; Liu Wei2; Li Hesheng3

(1. College of Materials Science and Engineering, Beijing University of Chemical Technology, Beijing 100029; 2. Beijing Key Laboratory of Membrane Science and Technology, College of Chemical Engineering, Beijing University of Chemical Technology, Beijing 100029; 3. Research Institute of Yanshan Petrochemical Co., SINOPEC, Beijing 102500)

Laccase was immobilized on the ceramic-chitosan composite support by using glutaraldehyde as the cross-linking reagent. The immobilization conditions and characterization of the immobilized enzyme were investigated. The immobilization of laccase was successfully realized when 3.0 mL of 1.25 mg/mL of laccase at a pH value of 4.0 reacted with 0.15 g of ceramic-chitosan composite support (CCCS) at 4 ℃ for 24 h. The immobilized enzyme exhibited a maximum activity at pH 3.0. The optimal temperatures for immobilized enzyme were 25 ℃ and 50 ℃. TheKmvalue of immobilized laccase for ABTS was 66.64 μmol/L at a pH value of 3.0 at 25 ℃. Compared with free laccase, the thermal, operating and storage stability of immobilized laccase was improved after the immobilization.

ceramic-chitosan composite support; immobilization; laccase; enzyme activity

1 Introduction

Laccase (E.C. 1.10.3.2 benzenediol: oxygen oxidoreductase) is a multi-copper oxidase, which is able to catalyze the one-electron oxidation of several aromatic substrates with the simultaneous reduction of dioxygen to two molecules of water[1]. The substrate range is fairly broad and includes polyphenols, polymethoxybenzenes, methoxy-substituted monophenols, benzenethiols, endocrine disrupting chemicals and other easily oxidizable compounds[2-3]. In particular, its effectiveness in wastewater treatment has been extensively reported[4]. Nevertheless, the laccase is often inactivated during its application process due to the wide variety of environmental conditions. Therefore, the improvements in the stability of laccase as well as the possibility of being reused in consecutive treatment cycles are the targets of considerable signifcance[5].

Enzyme immobilization technology is an effective method to promote enzyme reuse and to improve its stability. Laccases have been successfully immobilized on many different types of carriers, such as the activated carbon, the controlled porosity glass, the oxirane acrylic beads, the hydrophilic polyvinylidene fluoride microfiltration membrane and the magnetic chitosan microspheres[6].

Inorganic supports for enzyme immobilization are of great interest because of their durability, high mechanical strength for usage in packed-bed or fluidized-bed bioreactors, and relatively low cost[7]. If inorganic supports possess proper macrostructure such as the honeycomb monolith materials, the diffusion limitations of substrate transport toward biocatalyst are obviously minimized, and the effciency of substrate transporting process could be signifcantly run up[8-9]. The massive macroporous ceramics not only have all the advantages of inorganic supports, but also have the predominance of their shapes, which will be revealed in the design of packed-bed reactor and the separation of products[10]. We can expect that ceramics as the carrier for immobilizing enzyme will be widely used in the processes of industrial catalysis[11]. However,the binding capacity of ceramics with free enzyme is poor and the yield of enzyme immobilized through physical adsorption is low, so the inorganic support should be chemically modifed. We used chitosan as the second support to modify the ceramic material for the first time to our knowledge, and the result was satisfactory.

Chitosan [α(1→4)2-amino-2-deoxy-β-D-glucan] was chosen for the following reasons[12-16]. First, chitosan has good capabilities to form membranes on the surface of ceramics and higher recalcitrance to some microbial degradation than other natural supports, making itself an ideal carrier. Second, adsorption occurs through electrostatic interaction between the polycationic chitosan and the negative ions of laccase, which makes the procedure of immobilization easy. Third, we have discovered chitosan as an additive which can intensify the process of catalyzing oil by laccase in our previous study[17]. Fourth, chitosan is a product of deacetylation of chitin, which is the most abundant natural material on earth only after cellulose; thus, it is widely commercially available. However, there are still certain obstacles to its industrial applications. Directly using chitosan fakes or microspheres as supports, for instance, will cause a larger operation pressure and easy loss with the liquid phase[18]. The structure of internal pores would also exhibit a sterically hindered problem. If chitosan is prepared in the form of beads, it can entrap twice as much of the enzymes, as estimated according to the mass transported and the volume of reactive dye adsorbed onto the internal pores[19]. Chitosan beads have a density similar to water and are easily dispersed, and these characteristics make the operation rather inconvenient. This can be improved in conjunction with other solid powder to strengthen its physical properties, and thereby to expand its applications[20]. In a word, we choose chitosan as the second carrier coated on the surface of ceramic material, which can take full advantage of chitosan and overcome its defects.

Our previous study demonstrated that free laccase could catalyze oxidation of the emulsified and the dissolved oil contained in the oily wastewater. In this work, we further explored the method to immobilize laccase on the ceramic-chitosan composite support via crosslinking and physical adsorption. On the basis of these studies, we compared and characterized the immobilized laccase and free laccase in terms of their activity, thermal stability, and pH stability.

2 Experimental

2.1 Materials

Laccase was purchased from the Sigma-Aldrich Co. (America). The ceramic was provided by the Kaiming Catalyst Co., Ltd. (China). The ceramic material was cordierite, the main composition of which was Al2O3·SiO2·MgO. It was formed with square holes with a side length of 1.5 mm and a wall thickness of 0.4 mm. The bulk density of such ceramic was 700 kg/m3. The specific surface area of the ceramic was 24.60 m2/g as measured by the BET method. 2,2′-Azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) was purchased from the Appli. Chem. Co. (Germany). Chitosan (degree of deacetylation=90%) was obtained from the Sinopharm Chemical Reagent Co. (China). Glutaraldehyde (25%, v/v, aqueous solution) was purchased from the Tianjin Fuchen Chemical Reagent Co. (China). γ-Methylacryloyl oxypropyl trimethoxysilane (KH-570) was obtained from the Nanjing Xiangfei Chemical Research Institute (China). All other chemicals were of analytical reagent grade and no further purifcation was required.

2.2 Preparation of ceramic-chitosan composite support (CCCS)

The modifed solution was prepared by adding 0.26 mL of KH-570 into 30 mL of a mixture of anhydrous alcohol/ water (1:1, v/v) at a certain pH value (between 3.5 to 4.0). The ceramic was put into the modifed solution under stirring for 5 h at 30 ℃, and then washed with anhydrous alcohol and distilled water, respectively. The modifed ceramic was obtained after being dried overnight at 50 ℃. The chitosan solution was obtained by adding the chitosan powder into 1% (v/v) acetum solution under stirring for 10 min at room temperature. The modifed ceramics were impregnated with the chitosan solution for 1.5 h at 30 ℃, and then removed from the solution and dried overnight in a desiccator. The CCCS was obtained after the chitosan was coated on the modifed ceramic samples three times according to the same procedure mentioned above.

2.3 Immobilization of laccase

Firstly, one gram of CCCS was added into 25 mL of 6%glutaraldehyde solution for cross-linking under vigorous stirring for 15 min at 30 ℃. Then, it was washed by distilled water and dried overnight in a desiccator. Then, it was transferred into 5 mL of laccase solution with a concentration of 1.25 mg/mL for 24 h of immobilization at 4 ℃ and then was washed three times with the phosphate buffer.

2.4 Measurement of enzyme activity

Activity of the free and immobilized laccase was measured at 25 ℃ by an UV spectrophotometer (HACH DR5000) with 0.5 mmol/L of ABTS as the substrate (prepared with 0.2 M acetate buffer at a pH value of 3.0). The reaction mixture consisting of 8.0 mL of acetate buffer and 0.1 mL of free laccase solution (or 0.1 g of immobilized laccase) was added to 2 mL of substrate prior to being subject to reaction for 4 min at 25 ℃. During the process of reaction, the increase in the absorbance at 420 nm was measured. The molar extinction coefficient of ABTS was 36000 L/(mol·cm). One activity unit of laccase was defned as the amount of enzyme required to catalyze 1 μmol of substrate per minute[21-22].

2.5 Determination of optimal temperature and pH value

The thermal stability of laccase immobilized thereby was tested by incubating the immobilized enzyme at the specifed temperature (10℃—70℃) to determine the optimum operating temperature. The residual activity was calculated as the ratio of the activity at the optimum operating temperature. The pH stability of immobilized laccase was studied by incubating the immobilized enzyme at 25 ℃ in buffers of varying pH (3.0—8.0) and determining the activity at its optimum pH value. The relative activity was calculated as mentioned above and plotted against the pH value.

2.6 Kinetic properties

KmandVmaxvalues were determined by measuring the initial reaction rates of the free and immobilized laccase using different concentrations of ABTS as the substrate in a 0.1 mol/L tartaric acid buffer (pH=3.0) at 25 ℃.

2.7 Enzyme stability

Several consecutive operating cycles were performed by oxidizing ABTS in order to assess the operational stability of the immobilized laccase, which was washed three times with the acetate buffer. The procedure was repeated with a fresh aliquot of substrate, as described by Davis and Burns[23]. The storage stability was evaluated by storing the free and immobilized enzyme samples at 4 ℃ for one month.

3 Results and Discussion

3.1 Preparation of ceramic-chitosan composite support

3.1.1 Effect of KH-570 on chitosan coating

To determine the effect of γ-methylacryloyl oxypropyl trimethoxysilane (KH-570) on chitosan coated on ceramic, a series of experiments were conducted by putting the ceramic samples into the KH-570 solution with varying concentrations. The amount of residual chitosan on the support was assayed. The results are shown in Figure 1. The amount of residual chitosan on the supports increased with an increasing concentration of KH-570 when the concentration of KH-570 was lower than 2%. The amount of residual chitosan on the support reached a maximum value when the concentration of KH-570 was 2%. lt could be assumed that there was a chemical bonding reaction between KH-570 and the hydroxyl groups on the surface of ceramic[24]. The functional groups involved in the chemical bonding reaction can reduce the surface energy and enhance the surface bonding strength of ceramic, so chitosan can be frmly coated on the ceramic surface. The amount of residual chitosan on the support decreased with an increasing concentration of KH-570 in the range of 2%—10%. The reason could probably be that the KH-570condensation reaction would take place at the predominant position with an increasing concentration of KH-570. The coupling effect was rapidly decreased during this condensation reaction process.

Figure 1 Effect of KH-570 on chitosan coating

3.1.2 Characterization of ceramic-chitosan composite support (CCCS)

The FTIR spectra of the ceramic (a) and the ceramic-chitosan composite support (b) are shown in Figure 2. It can be seen from Figure 2(b) that the peak at 3 427 cm-1corresponds to the stretching vibrations of hydroxyl groups. The C-H stretching vibration of the polymer backbone is manifested through the strong peaks at 2 925 cm-1and 2 855 cm-1. The stretching vibrations of C—O bonds are found at 1 084 cm-1and 1 032 cm-1. The other bands are similar for both the ceramic and the ceramic-chitosan composite support. The existence of stretching vibrations of hydroxyl groups and C-H stretching vibration of the polymer backbone indicates that the chitosan coating was formed at the surface of ceramic.

Figure 2 FTIR spectra of ceramic (a), ceramic-chitosan composite support (b)

3.2 Immobilization of laccase

Before immobilization, the CCCS was placed in the phosphate buffer for 1 h. This process could make the chitosan molecule chains more relaxed in favor of the subsequent reaction of enzyme molecules with the active sites of CCCS. On the other hand, the swollen CCCS could make the diffusion of substrates easier and their contact with the immobilized enzyme more adequate[20].

An illustrative scheme of laccase immobilization on CCCS is shown in Figure 3. During the immobilization process, the impendent aldehyde groups on the surface of CCCS interacted with amino groups of enzyme to form formimino groups (-CH=N-). Since the properties of immobilized enzyme are signifcantly determined by the immobilization procedure, thus it is important to discuss the infuence of main factors on laccase immobilization.

3.2.1 Effect of glutaraldehyde on enzyme immobilization

The effects of concentration of glutaraldehyde ranging from 2% to 15% on the relative activity of immobilized enzyme are shown in Figure 4. The relative activity of immobilized enzyme increased with an increasing concentration of glutaraldehyde at the initial stage. The relative activity of immobilized enzyme reached a maximum at a glutaraldehyde concentration of 6%. The relative activity of immobilized enzyme began to decrease with an increasing concentration of glutaraldehyde. The reason could probably be that the CCCS cross-linked with a low concentration of glutaraldehyde had poor mechanical strength, easily leading to the falling of immobilized laccase from carriers[25]. With the increase of glutaraldehyde concentration, the amount of free aldehyde groups on the surface of the CCCS would increase and cause a higher laccase loading and activity. However, as the concentration of glutaraldehyde reached more than 6%, the extensive interaction of individual enzyme with aldehyde groups on the surface of the CCCS would change the enzyme conformation and cause a drop of immobilizedenzyme activity.

Figure 3 Schematic illustration of laccase immobilization and cross-linking with glutaraldehyde

The effects of cross-linking duration ranging from 5 min to 180 min on the activity of immobilized enzyme are shown in Figure 5. The relative activity of immobilized enzyme increased with the cross-linking duration at the initial stage. The relative activity of immobilized enzyme reached a maximum after 15 min. The relative activity of immobilized enzyme began to decrease with an increasing cross-linking duration. The first reason was that a longer cross-linking duration was propitious to the contact between aldehyde groups and amino groups of the CCCS. The second reason could probably be that the CCCS cross-linked in a short cross-linking duration had poor mechanical strength, easily leading to the falling of immobilized laccase from carriers. With the increase of cross-linking duration, the amount of free aldehyde groups on the surface of the CCCS would increase to cause a higher laccase loading and activity. However, as the cross-linking duration was longer than 15 min, the excessive interaction of aldehyde groups on the surface of supports would lead to a decreased number of active sites on the CCCS and cause the drop of immobilized enzyme activity.

Figure 4 Effect of glutaraldehyde concentration on enzyme immobilization

Figure 5 Effect of cross-linking durations on enzyme immobilization

It can be seen from the results depicted in Figure 4 and Figure 5 that the appropriate glutaraldehyde concentration was 6% and cross-linking time was 15 min.

3.2.2 Effect of pH value on enzyme immobilization

The laccase could be immobilized on the surface of the chitosan by electrostatic adsorption, whereas the pH value could influence the amount of enzyme adsorbed on the CCCS and its relative activity by altering the electronic properties of CCCS[26]. In order to evaluate the effect of pH value on laccase immobilization, laccase was immobilized on the CCCS at a certain pH values ranging from 3.0 to 8.0. The results are shown in Figure 6. The relative activity of immobilized enzyme decreased with an increasing pH value in the range of 4.0 to 8.0. The relative activity of immobilized enzyme reached a maximum at a pH value of 4.0. The possible reason could be that the chitosan coating could be partly dissolved and the enzyme would be denatured at lower pH value. This phenomenon could lead to a low amount of protein loading and laccase activity, so the relative activity of immobilized enzyme was low. The relative activity of immobilized enzyme decreased when the pH value was more than 4.0, because the laccase exhibited a lower stability under higher pH conditions.

Figure 6 Effect of pH value on enzyme immobilization

3.2.3 Effect of amount of added enzyme on enzyme immobilization

To determine the effect of amount of added enzyme on enzyme immobilization, the CCCS was put into the phosphate buffer at pH 4.0, which contained different concentrationsof laccase ranging from 0.25 mg/mL to 1.75 mg/mL. The activity of immobilized laccase was assayed, with the results shown in Figure 7. The relative activity of immobilized enzyme increased at frst and reached a maximum value of 1.25 mg/mL, and thereafter the relative activity of immobilized enzyme began to decrease with an increasing concentration of added laccase. It could be supposed that the amount of the active groups was defnite, and the quality of immobilized enzyme increased with an increasing concentration of laccase before the active sites of the CCCS were saturated. So the relative activity of immobilized enzyme was increased during this process. After the active sites of the CCCS were saturated, more enzyme molecules could enter the interior of CCCS, which would prevent the enzyme molecules from effciently performing the catalytic capability because of the limitation on substrate diffusion[27]. Meanwhile, the enzyme could be hidden and some active sites were damaged in the highly immobilized enzyme system.

Figure 7 Effect of added enzyme concentration on enzyme immobilization

3.2.4 Effect of temperature on enzyme immobilization

In order to evaluate the effect of temperature on laccase immobilization, laccase was immobilized on the ceramic-chitosan composite support cross-linked with glutaraldehyde at different temperatures ranging from 0 ℃ to 30 ℃. The activity of immobilized enzyme was assayed. The results are illustrated in Figure 8. The relative activity of immobilized enzyme was increased with an increasing temperature at the initial stage. The relative activity of immobilized enzyme began to decrease when the temperature increased from 4 ℃ to 30 ℃. The relative activity of immobilized enzyme reached a maximum value at 4 ℃. It is assumed that increase of temperature could accelerate the velocity of the enzyme molecules in the low temperature region (at 4 ℃), so more enzyme molecules were immobilized on the CCCS. When the temperature was higher than 4 ℃, the increase in temperature could accelerate the rate of enzyme denaturation. So the relative activity of immobilized enzyme began to decrease.

Figure 8 Effect of temperature on enzyme immobilization

3.2.5 Effect of time on enzyme immobilization

Laccase was immobilized on the ceramic-chitosan composite support over different time ranging from 3 h to 48 h. The effect of immobilization duration on the immobilized laccase activity is shown in Figure 9. The relative activity of immobilized enzyme was increased with an increasing immobilized duration between 3 h to 24 h. The relative activity of immobilized enzyme began to decrease with an increasing immobilization duration. The relative activity of immobilized enzyme reached a maximum at 24 h. Under the conditions mentioned above (when 1.25 mg/mL of laccase in phosphate buffer at pH 4.0 reacted with the ceramic-chitosan composite support at 4 ℃ for 24 h), a 51% yield of enzyme coupling was obtained and its activitywas found to be 55.87 U/g-support. The results indicated that longer immobilized duration was conducive to the contact between active groups and enzyme, before the active sites of the CCCS were saturated. After the active sites of the CCCS were saturated, more enzyme molecules entered the interior of CCCS. The excessive immobilized enzyme could make the apertures of carriers become thin, leading to a reduced accessibility to the active sites on the substrate[28].

Figure 9 Effect of time on enzyme immobilization

3.3 Properties of free and immobilized laccases

3.3.1 Effect of temperature on laccase activity

The effect of temperature on the activity of free and immobilized laccase is shown in Figure 10. The relative activity of immobilized enzyme and free enzyme reached a maximum at 25 ℃ and 50 ℃, respectively. The reason for the appearance of two peaks might be attributed to the unstable property of laccase at low temperature coupled with low pH value, which caused the change of catalytic capability of enzyme in the low temperature region[20]. On the other hand, the relative activity reached above 80% in the temperature ranging from 15 ℃ to 70 ℃ for free laccase and from 20 ℃ to 35 ℃ for the immobilized laccase, respectively. The relative activity of free enzyme displayed a broader tolerance range to heat than the immobilized enzyme. The results indicated that the higher activity of immobilized enzyme became more dependent on the temperature.

Figure 10 Effect of temperature on activity of the enzyme

3.3.2 Effect of pH value on laccase activity

The effect of pH value on the activity of the free and the immobilized enzyme is shown in Figure 11. Similar trend of pH effect on the activity of the free and the immobilized laccase was obtained in the pH range studied (3.0—6.0). The immobilized laccase exhibited a higher enzyme activity, as compared with the free laccase. The result could assumed that the micro-environment of the immobilized enzyme and the bulk solution usually showed unequal partitioning of H+and OH-concentration due to their electrostatic interaction with the matrix, which could often lead to the displacement in the pH activity profile[29-30]. This analogous result has been observed by other investigators[20-31]. Free enzyme has a low activity, which might be caused by the reduced stability of free laccase at lower pH value.

Figure 11 Effect of pH on activity of the enzyme

3.3.3 Reuse and storage stability of immobilized laccase

The operating stability of the immobilized laccase in the current study was evaluated in a repeated batch process. The effect of repeated use on the activity of immobilized laccase is shown in Figure 12. The immobilized laccase retained an activity of 90.6% after 10 cycles. The main reason for the loss of activity was ascribed to the leakage of protein from the ceramic-chitosan composite supports.A relative activity retention rate equating to 85% after 10 cycles and reaching 65% after 4 cycles was reported by Alessandro[26]and Magnan[32], respectively.

Figure 12 Operating stability of immobilized enzyme

The storage stability of free and immobilized laccase is shown in Figure 13. The activity of the immobilized laccase was reduced by 5.4% after 30 days at 4 ℃. Under the same condition, the activity of free laccase dropped to 94.2% after 30 days. The study result showed that the storage stability of the immobilized laccase was higher than the free laccase.

Figure 13 Storage stability of free and immobilized enzyme

3.3.4 Kinetic properties

The kinetic parameters of the free and the immobilized laccase samples in terms ofKmandVmaxare shown in Table 1. The process in which laccases were immobilized on the CCCS led to a decreasedVmaxvalue of 0.036 μmol/(L·min) as compared to 0.223 μmol/(L·min) for the free laccase. TheKmvalue of immobilized laccase (66.64μmol/L) was 1.9 times higher than that of free laccase (34.41 μmol/L), denoting that the immobilized laccase on the CCCS had lower affnity towards the substrate. A higher affnity of the immobilized laccase was obtained than that reported by other investigators. The increase inKmvalue might be caused by the lower accessibility of the substrate to the active sites on the support, or the loss of enzyme fexibility necessary for substrate binding, or the diffusion limitations on substrate and products during the immobilization procedure.

Table 1 Kinetic parameters of free and immobilized laccase

4 Conclusions

In this work, we developed a novel ceramic-chitosan composite support as the enzyme immobilization carrier. Laccase was immobilized on the support surface by using glutaraldehyde as the cross-linking agent. The optimal process for immobilization of laccase was successfully realized when 3.0 mL of solution consisting of 1.25 mg/mL of laccase in phosphate buffer (0.1 mol/L, pH=4.0) reacted with 0.15 g of ceramic-chitosan composite support at 4 ℃ for 24 h. Under this condition, a 51% yield of enzyme coupling was obtained, with its activity equating to 55.87 U/g-support.

The immobilized enzyme exhibited a maximum activity at a pH value of 3.0. The optimal temperature suited to the immobilized enzyme was 50 ℃. TheKmvalue of immobilized laccase for ABTS was 66.64 μmol/L in a pH-3.0 buffer at 25 ℃, which was higher than that of free enzyme (34.41 μmol/L), denoting that the immobilized laccase had lower affnity towards the substrate. In addition, the immobilized laccase exhibited remarkably improved stability properties as evidenced by various parameters, such as the high pH stability, an activity retention rate of 90.6% after 10 operating cycles, and an activity retention rate of 94.6% after 30 days of storage.

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Received date: 2016-07-29; Accepted date: 2016-10-15.

Professor Liu Wei, Telephone: +86-10-64453979; E-mail: liuw@mail.buct.edu.cn, lizl@mail.buct. edu.cn.