Enhancing laccase stability and activity for dyes decolorization using ZIF-8@MWCNT nanocomposite

Pascal Habimana,Yanjun Jiang,Jing Gao,Jean Bernard Ndayambaje,Osama M.Darwesh,Jean Pierre Mwizerwa,Xiaobing Zheng,*,Li Ma,*

1 School of Chemical Engineering and Technology,Hebei University of Technology,Tianjin 300130,China

2 University of Rwanda,College of Science and Technology,3900 Kigali,Rwanda

3 Department of Agricultural Microbiology,National Research Centre,Cairo 12622,Egypt

4 College of Mechatronics and Control Engineering,Shenzhen University,Shenzhen 518060,China

Keywords:Metal-organic framework Zeolitic imidazolate framework Reusability and stability Immobilized laccase Dye decolorization

ABSTRACT The continuous use of chemical dyes in various industries,and their discharge into industrial effluents,results in severe problems to human life and water pollution.Laccases have the ability to decolorize dyes and toxic chemicals in industrial effluents as green biocatalysts.Their possible industrial applications have been limited by poor reusability,low stability,and loss of free laccase action.In this research,laccase was immobilized on zeolitic imidazolate framework coated multi-walled carbon nanotubes(Laccase@ZIF-8@MWCNTs)via metal affinity adsorption to develop an easy separable and stable enzyme.The optimum reaction conditions for immobilized laccase are at a pH of 3.0 and a temperature of 60 °C.The immobilized laccase was enhanced in storage and thermal stability.The results indicated that Laccase@ZIF-8@MWCNTs still maintained 68% of its original activity after 10 times of repeated use.Most importantly,the biocatalytic system was applied for decolorization of different dyes (20 mg·L-1)without a mediator,and up to 97.4% for Eriochrome black T and 95.6% Acid red 88 was achieved in 25 min.Biocatalysts with these properties may be used in a variety of environmental and industrial applications.

1.Introduction

The possibility of environmental contamination has been increased rapidly due to the use of chemical dyes in various industries such as paper printing,pharmaceutical,cosmetics,clothing,rubber,leather,color photography,and other industries activities[1].Dyes discharged from these industries lead to severe human problems(mutagenic,carcinogenic,and teratogenic)or water pollution.Besides,with our naked eyes,the presence of 1 mg·L-1of dyes in water can be observed and reduces light penetration through the water surface,thus preventing aquatic flora photosynthesis [2].In the textile or other coloring industries,Acid Red 88 and Eriochrome Black T are among the dyes used in various materials such as textiles,nylon,leather,silk,and wool.Meanwhile,their degradation products are poisonous,mutagenic,and carcinogenic to human life [3].Moreover,due to their reactive stability and high solubility in water,it is extremely hard to remove dyes from industrial wastewater [1,3].

Conventional wastewater treatment techniques such as adsorption,photocatalytic degradation,and flocculation have been studied in detail [1],it has also been documented that some of these techniques require higher costs and are difficult to operate,as well as generating secondary contaminants [1,4].Environmentally friendly and safe approaches like enzymatic biocatalysis are therefore essential for the treatment of dye-containing industrial effluents[5,6].Free enzymes,however,suffer from different disadvantages,including low stability,non-repeatable utilization,and high costs,which are the feature limitations of their functional application[7].Therefore,enzyme immobilization on suitable support can remove these limitations and making the enzyme more commercially and industrially feasible [4,7].Emerging this technology,the enzyme might be immobilized on various support by chemical or physical techniques to produce an insoluble enzyme,which displays enhanced storage and thermal stability,as well as good reusability;such immobilized enzyme possesses potentials in industrial and environmental applications [5].

Various enzyme immobilization methods have been thoroughly examined,including crosslinking,covalent binding,adsorption,and entrapment [8,9].Entrapment is known to be the best option since it is a moderately terrible process that provokes little destruction to the enzyme’s native structure.Even though this method is inexpensive and facile,the carrier can swell and cracks easily,resulting in enzyme and cell leakage from the support,which reduces immobilization efficiency and enzyme activity[8,9].The covalent binding method is defined by the strong covalent bond formed between the enzyme molecules and supports.Besides,this method can be employed to preclude enzyme leakage,and glutaraldehyde is mostly used as support activation or spacer arms to provide a strong connection between the support and enzyme,which results in improved operational stability [8,9].However,due to the harsh reaction conditions and complicated operations in the immobilization process,it is facile to cause conformational flexibility of the enzyme molecule,thus,destroying its active site,and it is difficult to ensure a high recovery rate of enzyme activity [8-10].

The adsorption method is simple,cheap,and no other functional groups are required to modify the carrier resulting in less destruction of the enzyme’s structure.However,due to the weak attraction between the support and enzyme molecule,the enzyme molecule can be easily lost during practical use,leading to the low operation stability of enzyme and product contamination [8-10].By crosslinking,the enzymes are strongly bound to the suitable carrier by a crosslinker comprising two or more functional groups.The crosslinker may augment the strength of the support by creating a spatial structural grid.Glutaraldehyde is widely employed as a crosslinker,which comprises two aldehyde groups where some aldehyde group of this reagent bind with the support and the remaining react with the amino group on the enzyme molecule.The interaction between the substrate and enzyme is firm,leading to good reusability and stability.Difficulty to control the reaction process,denaturation of enzyme structure,and use of toxic reagents are the main disadvantages of this method [10-12].

Selecting an appropriate enzyme carrier can therefore play a significant role in all enzyme immobilization techniques that can assist the enzyme immobilization process,improve catalytic performance,reusability,high enzyme loading,and increase stability[9-12].Nanomaterials such as metal-organic frameworks (MOFs),nanoparticles,protein-metal hybrid nanoflowers,nanotubes,nanosheets,and nanofibers,have commonly been utilized as support materials for immobilizing enzyme due to their fewer diffusion limitations and larger specific surface areas [13-16].Enzyme immobilization through proteins as organic and metals as inorganic components reveals variable impacts on enzyme features.Generally,nucleation,aggregation,and anisotropic are the three steps followed during the synthesis of inorganic-protein hybrids as a flower-like structure.Due to their suitable confining environments,large surface area,and porous nature,these nanoflowersbased systems have been considered as appropriate support for immobilizing enzymes.The lower stability,long incubation period,and poor reusability as the results of their soft nature are the disadvantages of the nanoflower-based enzyme immobilization [16-19].To improve the reusability and stability of the system,Patelet al.[19] immobilized laccase with Cu ion-based proteininorganic hybrid through glutaraldehyde as cross-linker,and the biocatalytic systems developed exhibited significant enhancement in the catalytic features of laccase,as well as high reusability and stability.

Due to their high chemical stability,low price,efficient enzyme loading,strong biocompatibility,and high surface area,carbon nanotubes have been widely employed as a support material for enzyme immobilization [7,20-23].However,their practical industrial application in bioremediation is hindered by its aggregation abilityviathe strong interaction between the tiny particles of carbon,resulting in a smaller surface area [7,23].Besides,the carbon nanotubes have poor dispersibility and solubility characteristics in an aqueous solution.Therefore,carbon nanotubes may be synthesized by modifying their surface to reduce aggregation and augment the accessibility of the surface area [24,25].Metal-organic frameworks (MOFs) are a fascinating group of porous crystalline materials constructed from organic linkers and metal ionsviacoordination attractions [13].Due to their large porosity,tuned functionality,and high surface area,MOFs have recently been studied for their practicability for several uses[13-15].Zeolitic imidazolate frameworks (ZIFs),in specific ZIF-8,which consists of imidazolate linkers,and metal ions,superior chemical and thermal stability,maximal enzyme loading,low cytotoxicity,fewer diffusion limitations,and easy preparation process,were studied extensively for enzymes immobilization [15,16,26].

Most notably,different enzymes were immobilized on or inside metal-organic frameworks where storage stability,multi-cycle reusability,and thermal stability were significantly improved[13].The close relation between enzymes and MOFs that preclude enzyme leaching from the pores is the product of these enhanced properties [13,14].However,the preparation of metal-organic framework-based nanocomposites employing appropriate support materials is highly needed.Carbon nanotubes (CNTs) are suitable materials for the synthesis of metal-organic framework nanocomposites[27,28].Therefore,based on the aforementioned properties,zeolitic imidazolate framework coated multi-walled carbon nanotubes (ZIF-8@MWCNT) was prepared,characterized by X-ray photoelectron spectroscopy (XPS),scanning electron microscopy(SEM),Brunauer-Emmett-Teller(BET),and Fourier transform infrared spectroscopy (FTIR) and then used in enzyme immobilization process.

Laccases as an oxidoreductase enzyme was chosen as a model due to their capacity to oxidize a wide variety of inorganic and organic substrates with concomitant release of water molecules,besides laccases,have a broad substrate specificity towards phenolic and non-phenolic compounds [29,30].Most notably,these enzymes are used in the dyeing,printing,and paper industries,as well as in the decolorization of industrial dyed effluents[6,30].However,instability of free laccase is still a problem due to long-term exposure of this enzyme to organic solvents [31].Therefore,laccase immobilized on some supports can solve this problem.Thus,in this work,the ZIF-8@MWCNT nanocomposite was synthesized from Zinc nitrate salt,2-methylimidazole,and multi-walled carbon nanotubes as raw materials,meanwhile the laccase enzyme was immobilizedviametal affinity adsorption process.The obtained biocatalyst system was named Laccase@ZIF-8@MWCNT.

Laccase has gotten a lot of attention as a result of its use in the treatment of wastewater from the textile and paper industries.When the laccase mediator method was used in continuous bioreactors,however,the cost was a major issue because the redox mediator had to be added regularly.When the redox mediator is missing,the reaction takes longer [32].Othmanet al.[33],for example,documented the discoloration of Reactive Black 5 by immobilized laccase with multi-walled carbon nanotubes as a carrier and found that it took 24 h to remove 84 percent color.On the other hand,Misraet al.[34] used laccase immobilized with epoxy activated polyethersulfone beads as a carrier,showing that the discoloration efficiency of the dye was 88 percent in 10 days.In another study,Jaiswalet al.[35] used papaya laccase immobilized with chitosan to decolorize Indigo carmine,showing 100% efficiency of color removal in 8 h.Yuanet al.[36]investigated the discoloration of Reactive Blue 19 employing immobilized laccase with bacterial nanocellulose (BNC) as support,the result displayed that the decolorization efficiency of the dye in 5 h was 88.2%.Also,dyes decolorization by laccase enzyme depends on the dye’s structure,as the mechanism of decolorization differs accordingly [33,36];Furthermore,all of the dyes tested took longer than 4 h to be decolorize effectively.However,decolorization of azo dyes by immobilized laccase without the use of a mediator is difficult to achieve,necessitating the development of a more efficient process that can decolorize azo dyes in less time.

In the current study,the ZIF-8@MWCNT nanocomposite was selected as a suitable carrier to immobilize the laccase enzyme,and various preparation parameters of Laccase@ZIF-8@MWCNT were optimized,as well as the stability of Laccase@ZIF-8@MWCNT was also investigated.In the discoloration of Acid Red 88 and Eriochrome Black T without a mediator,the Laccase@ZIF-8@MWCNT obtained was finally used as a biocatalyst.The results displayed that the Laccase@ZIF-8@MWCNT was capable to decolorize all dyes,which provides a novel efficient method in industrial and environmental bioremediation.

2.Experimental

2.1.Materials

Laccase(120 U·g-1)was purchased from Shanghai Yuan Ye Bio-Technology Co.,Ltd(China),2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS,98%) and multiwalled carbon nanotubes(MWCNT) with a mean diameter 5-50 nm were purchased from Aladdin Biochemical Technology Co.,Ltd(Shanghai,China).Methanol and 2-methylimidazole(2-MeIM)were purchased from Sigma-Aldrich.Zinc nitrate hexahydrate (Zn (NO3)2·6H2O,99.99%) was purchased from Aladdin Industrial Corporation.Acid Red 88(AR88) and Eriochrome Black T were purchased from Hubei Jusheng Technology Co.,Ltd (China).Without further purification,all reagents were used as obtained.

2.2.Synthesis of carboxyl-functionalized MWCNTs

The reported methods with some modification were employed to synthesize carboxyl-functionalized MWCNT [37,38].Typically,a mixture comprising HNO3/ H2SO4(1:3 v/v),100 ml were added into 500 mg of MWCNTs and ultrasonicated at 40 °C for three hours.Subsequently,at 8000 r·min-1the mixtures were centrifuged for ten minutes,washed,and the sediments (MWCNTCOOH) were collected,finally dried at 60 °C.

2.3.Synthesis of ZIF-8@MWCNT

The zeolitic imidazolate framework coated multiwalled carbon nanotubes (ZIF-8@MWCNTs) were prepared based on the published literature with some modifications [27,28].120 mg of carboxyl-functionalized MWCNT and 0.649 g 2-methylimidazole were dissolved into 20 ml methanol and ultrasonically mixed for 30 min at room temperature.Then,a solution containing 0.2933 g Zn (NO3)2.6H2O and 20 ml methanol was added directly to the previously prepared dispersion at room temperature with intense stirring for 30 min.The mixtures obtained were eventually moved to a high-pressure reactor and heated for six hours at 90°C.The resulting mixtures were obtained by a centrifuge,washed with methanol,and dried at 60 °C.

2.4.The optimization of laccase immobilization

To optimize the immobilization parameters,various concentrations of laccase (3,4.5,6,7.5,and 9 mg·ml-1) prepared in sodium acetate buffer with 0.2 mol·L-1and pH 4.0 were separately added to 10 mg of ZIF-8@MWCNT,and the adsorption process was then performed at 25 °C for 1.5 h.The adsorption time was optimized by dissolving an optimum concentration of laccase enzyme prepared in sodium acetate buffer (0.2 mol·L-1,pH 4.0) into 10 mg of ZIF-8@MWCNT and the resulting mixtures were shaken in a water bath shaker at 25 °C for various time intervals 0.5-2.5 h.After centrifugation,the obtained precipitates were washed with sodium acetate buffer (0.2 mol·L-1,pH 4) and then kept at 4 °C after freeze-drying.The immobilized enzyme activity was calculated by taking starting enzyme activity (free enzyme activity)minus supernatant enzyme activity (after immobilization),and the maximum activity at each step was considered as 100% and then the relative activities of the immobilized enzyme were calculated accordingly [5,15].The optimal adsorption time or laccase concentration was determined accordingly.

2.5.Assay of enzyme activity

Under assay conditions,one unit of laccase activity represents the amount of enzyme that converts one μmol of ABTS per min[6,15,39].The laccase activity was quantified after incubation of the 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) ABTS(1.8 ml,1.2 mmol·L-1) solution prepared in buffer solution(0.2 mol·L-1,pH 4)with the free laccase(0.2 ml)at 30°C for about 4 min.The laccase activity was tested spectrophotometrically at 420 nm.

At 30°C,10 mg Laccase@ZIF-8@MWCNT,sodium acetate buffer(1 ml,0.2 mol·L-1,pH 4),and ABTS (1 ml,1.2 mmol·L-1) were mixed and magnetically stirred for 4 min.Samples were immediately filtered,and the activity of the laccase was spectrophotometrically measured at 420 nm.The 36.0 mmol·L-1·cm-1was used to assess the activity of both enzymes as the molar extinction coefficient of ABTS.Finally,U·ml-1was used as a unit for the free operation of laccase,whereas U·mg-1represented the immobilized laccase.All experiments were carried out three times separately,and relative activities were calculated accordingly.

2.6.Characterization of the produced materials as well as immobilized laccase

The morphologies of the as-synthesized pristine MWCNT,MWCNT-COOH,and ZIF-8@MWCNT were investigated by the SEM employing Nova Nano SEM 450 field-emission microscope.XPS spectra of ZIF-8@MWCNT were recorded by a Thermo Scientific Al K-Alpha X-ray photoelectron spectrometer.The functional group compositions of MWCNT-COOH,ZIF-8@MWCNT,and Laccase@ZIF-8@MWCNT were examined by FTIR.The nitrogen adsorption/desorption isotherms were recorded at 77 K with a Quantachrome instruments version 11.04.The BET method was employed to investigate the specific surface of the produced samples.The size distribution and pore volumes of the produced samples were examined utilizing the Barrett-Joyner-Halenda (BJH)model.

2.7.Effect of pH values and temperature against laccase activity

To evaluate the effect of pH values and temperature against laccase activity,the free laccase or immobilized form was incubated in various pH values (2.0-8.0) at 30 °C or different temperatures(30-70 °C) in optimum buffer solution (0.2 mol·L-1,pH 3.0).The activities of the immobilized and free laccase at a chosen range of temperature and pH were examined respectively.

2.8.Thermal and pH stability of laccase

To test the pH stability,the immobilized and free laccase were dissolved in various buffers (pH 3 and 8,0.2 mol·L-1) at 30 °C for variable time intervals.Relative activities of the free laccase and immobilized form at a chosen temperature and pH were examined,respectively.Similarly,the thermal inactivation stability was tested after incubating immobilized or free laccase in phosphate buffer solution (PBS,0.2 mol·L-1and pH 3.0) at 65 °C for 180 min,and then after the relative activity was tested to determine the temperature stability.

2.9.Storage stability of laccase

To study the stability of the enzyme in storage,the Laccase@ZIF-8@MWCNT and free laccase were kept in buffer solution (PBS,0.2 mol·L-1,pH 3.0) at 4 °C for 25 days.Sampling was done at every 5 days interval,and the laccase activity at optimal pH on day zero was set as 100%.

2.10.Reusability of immobilized laccase

To further examine the reusabilities,the laccase immobilized on Zeolitic imidazolate frameworks coated multi-walled carbon nanotubes (Laccase@ZIF-8@MWCNT) was utilized in the reaction for a specified number of cycles(4 min each)using 1.2 mmol·L-1ABTS as substrate prepared in buffer solution (pH 3.0,0.2 mol·L-1) at 60 °C.The Laccase@ZIF-8@MWCNT activity was evaluated at 30 °C.After each laccase activity measurement,the Laccase@ZIF-8@MWCNT was obtained by centrifuge and washed with buffer solution (pH 3.0,0.2 mol·L-1) before being employed in the next reaction.

Decolorization kinetics and decolorization reusabilities of the azo dyes were studied without a mediator.The ZIF-8@MWCNT,Laccase@ZIF-8@MWCNT,or free laccase was separately added to(20 mg·ml-1,20 ml)of Acid Red 88 or Eriochrome Black T prepared in buffer(0.2 mol·L-1,pH 3.0),respectively.The reaction processes were accomplished at 60 °C with stirring for different time intervals 5-25 min for the decolorization kinetics while decolorization reusabilities were carried out for 25 min(each reaction).In details,10 mg of the biocatalytic systems (Laccase@ZIF-8@MWCNT,0.918 U),ZIF-8@MWCNT (10 mg) or 6 mg·ml-1free enzyme (5 ml,43 U·ml-1),and dyes (20 ml) were mixed in one neck round flask(100 ml)separately for each dye.At particular time intervals,a certain quantity of samples was taken,filtered.Subsequently,using spectrophotometrically,the absorbance was analyzed through various wavelengths of the employed dye Acid Red 88 (505 nm) and Eriochrome Black T (523 nm).The decolorization percentage was measured as follows:

WhereDis the percentage decolorization,B0is the beginning absorbance,andBtis the final absorbance of the dye at each time interval.

3.Results and Discussion

3.1.Characterization of the produced materials

The scanning electron microscopy(SEM)images were collected to examine the changes in the structure and surface morphologies of MWCNT,MWCNT-COOH,and ZIF-8@MWCNT.The obtained results showed that MWCNT before and after functionalization with carboxylic groups were nanotubular in shape with an average size of 500 and 1000 nm,respectively(Fig.1(a)and(b)).However,the tubes observed after functionalization were shorter and with a more dense structure,which further confirmed that the acid functional groups have been successfully attached to the surface of carbon nanotubes.Similar trend results were reported by previous studies [7,21].Additionally,the SEM images of ZIF-8@MWCNT nanocomposites were illustrated in Fig.1(c)from which the results indicated that the ZIF-8 nanocrystals were surrounded on the surface of MWCNT,and the as-synthesized ZIF-8@MWCNT nanocomposites exhibited both properties of carbon nanotubes and ZIF-8 metal-organic frameworks.It has been reported that,when the quantity of multiwalled carbon nanotubes is low,most of them are enclosed with the ZIF-8 nanocrystals,and with augmenting ZIF-8 nanocrystals,the structural shape of ZIF-8@MWCNT nanocomposites looks like a bunch of grape [27,28,40].

Fig.1.The morphology of obtained samples.SEM images of (a) MWCNT,(b)functionalized MWCNT (MWCNT-COOH) and (c) ZIF-8@MWCNT.

The chemical state and elemental composition of ZIF-8@MWCNT were further analyzed by XPS.The signal peaks of O,Zn,N,and C elements could be displayed in the survey spectrum of ZIF-8@MWCNT (Fig.S1(a),Supplementary Material).Fig.S1(be) illustrated the comparison data of the XPS of O 1s,Zn 2p,N 1s,and C 1s spectra.The Zn 2p broad signals are found at 1043.98 and 1021.3 eV,which are well attributed to the Zn 2p1/2 and Zn 2p3/2 intensity spectrum of the Zn2+oxidation state [26-28,41].The obtained results proved the successful synthesis of ZIF-8@MWCNT.

The porosity of ZIF-8@MWCNT was investigated by nitrogen adsorption/desorption isotherms.The obtained results displayed that the nitrogen adsorption/desorption isotherms of ZIF-8@MWCNT nanocomposites exhibited a typical type IV and I isotherms (Fig.2(a)),which indicates the presence of microporous and mesoporous structures with various pore size in ZIF-8@MWCNTs [42,43].The pore volume and BET were examined to be 0.714 cm3·g-1and 328.778 m2·g-1,respectively.The pore size distribution was investigated by BJH.Fig.2(b),indicated that ZIF-8@MWCNT had an average pore size of 4.149 nm,which could offer a substantial amount of sites for enzyme attachment,which was beneficial for enzyme immobilization.

To investigate the surface properties of functionalized MWCNT,ZIF-8@MWCNT,and Laccase@ZIF-8@MWCNT,FTIR analysis was carried out.As can be observed in(Fig.3),the two peaks at around 2930 and 2858 cm-1were assigned to the asymmetric and symmetric of C-H stretching.The signal peaks in the range between 1573-1683 cm-1for curves a,b,c,and d are assigned to the aromatic carbon-carbon double bond,and the peaks at around 3440 cm-1for curves a,b,c,and d are ascribed to the-OH groups.However,the intensity peak of -OH groups for curve b was reduced,confirming the successful coating of Zn2+on MWCNT[27,28].The intensity absorption peaks at around 1422-990 cm-1for curves b and c are attributed to the imidazole ring.Additionally,the peaks at 1303-990 cm-1are assigned to the stretching vibration of C-O,thus,confirming the existence of oxygen on the surface of MWCNT[28].Most importantly,the amide I and amide II peaks of the secondary structure of laccase were occurred at 1630 and 1422 cm-1for curve c which confirmed the successful attachment of laccase to ZIF-8@MWCNT [39,44].

Fig.2.(a) Nitrogen adsorption-desorption isotherm at 77 K and (b) pore size distribution profile of ZIF-8@MWCNT nanocomposites.

Fig.3.FTIR spectra of (a) MWCNT-COOH,(b) ZIF-8@MWCNT,(c) Immobilized laccase,and (d) Free laccase.

3.2.The optimization of laccase concentration and adsorption time

In this study,the effect of the intial concentration of laccase against the activity of laccase immobilized on ZIF-8@MWCNTviametal affinity adsorption was studied.As can be displayed in Fig.4(a),when the laccase concentration augmented from 3 mg·ml-1to 6 mg·ml-1,the ZIF-8@MWCNT immobilized laccase relative activities were also augmented.The optimal activity of the ZIF-8@MWCNT immobilized laccase was achieved when the concentration of laccase was (6 mg·ml-1,45.93 U·ml-1).Further increase in the concentration of laccase from 6 mg·ml-1to 9 mg·ml-1,the activity of the immobilized enzyme starts to decrease.These could be the reasons:Firstly,when the concentration of enzyme is comparatively low,the laccase may not adequately be adsorbed onto the ZIF-8@MWCNT support,resulting in the low amount of immobilized enzyme and thus,low enzyme activity occurred.Secondly,laccase’s overcrowded accumulation altered its conformational stability,and some active centers were destroyed or blocked,resulting in the reduction of the laccase activity and easily subjected to the surrounding terrible microenvironment[45,46].Thus,6 mg·ml-1of the laccase concentration was adopted in this study.

The relative laccase activity of Laccase@ZIF-8@MWCNT can change along with increasing the immobilization time.Laccase’s relative activity is time-dependent,increasing as immobilization time increases (Fig.4(b)).Herein,the highest relative activity of the laccase immobilized on ZIF-8@MWCNT was achieved after 1 h of immobilization.On the contrary,the laccase’s relative activity reduced after 1 h.These could be the reasons:the long-term shaking can trigger the laccase denaturation,or the overcrowded loading of laccase on ZIF-8@MWCNT alters its conformational stability,thus,leading to a decrease in enzyme activity [45,47].

3.3.Effect of pH values and temperature against laccase activity

Due to the ionization shift of enzyme functional groups,the pH can alter the behavior of enzyme activity in the reaction process[4].Also,the optimal pH value for laccases is highly dependent on the substrate type and redox potential.Moreover,fungal laccases have an optimal pH value at an acidic condition,and the pH stability varies greatly depending on the enzyme’s origin[29,48].In this study,the influence of pH against the laccase activity was carried out at the pH value ranging from (2-8).As displayed in Fig.5(a),the highest relative enzyme activity has occurred at pH 3.0 for both immobilized and free laccase with higher relative activity for the immobilized laccase,which is in line with the previously reported studies [34,49,50].Also,the activity of Laccase@ZIF-8@MWCNT on both alkaline and acidic medium continued to be higher than free laccase,which might be due to its stability.Although the Laccase@ZIF-8@MWCNT maintained 45% relative activity in the pH range of 4-7,the free laccase kept 28%of its highest relative activity.Similar findings were published[6,8].However,in alkaline media,the relative activity of both free laccase and immobilized form was reduced,but slightly for the immobilized laccase.This might be due to the increase of hydroxyl ions which tend to connect to the type 2 or type 3 sites of the enzyme limiting the transfer of electrons from type 1 to type 2 or type 3 sites.A Similar trend in pH decrease in activity was reported [34,49].Thus,the free laccase activity decreased along with augmenting pH,and about 15% of the relative activity was maintained at pH 8.0 for free laccase while immobilized laccase retained 35%.Based on these findings,the immobilized laccase is more resistant to alkaline and acidic media,which is advantageous for a wide range of applications.

Fig.4.(a)Effect of initial laccase concentration on the activity of the immobilized enzyme.Immobilization was carried out at 25°C for 1.5 h at varying laccase concentrations and (b) adsorption time on the activity of the immobilized enzyme.Immobilization was carried out at 25 °C for different time intervals with a laccase concentration of 6 mg·ml-1.Laccase activities were measured using 1.2 mmol·L-1 ABTS (1 ml) as a substrate at 30 °C under pH 4.0.

Fig.5.Effects of (a) pH value and (b) temperature on the activities of laccase.(c) pH and (d) thermal stabilities of free and immobilized laccase.

Temperature is a crucial factor to regulate during the enzyme immobilization process since it can significantly affect the enzyme’s activity [4,49].The effect of temperature against the behavior of free laccase and immobilized form was tested in this work.As displayed in Fig.5(b),with an augment in temperature,the activity for both laccases increased along with augmenting the temperature from 30 to 60°C,but with further increase in temperature from 60 to 70 °C,both laccases’ activity diminished but only slightly for immobilized laccase.60 °C was found to be the best temperature for both immobilized and free laccase.In comparison to free form,laccase immobilized on ZIF-8@MWCNT is more stable at high temperatures.This is in line with the works reported,which could be supported for the following reasons:The temperature rise is beneficial for the strong laccase conjugate to be assisted by the adsorption of metal affinity,which could trigger the molecular shift of the enzyme to high activity.A further increase in temperature,on the other hand,may result in the loss of laccase activity due to denaturation of the enzyme structure[45,51].

The external microenvironment,particularly terrible conditions(pH,temperature,etc.)that may destruct enzyme structures,could have a significant impact on enzyme activity [34,48].The stability of free laccase and its immobilized form under various pH values was investigated in this study.Laccase was incubated at different pH (pH 3.0 and 8.0) for different time intervals and the activities were also tested.As displayed in Fig.5(c),at pH 3.0,free laccase retained 82%of its beginning activity at 30°C for 4 h,while the laccase immobilized on ZIF-8@MWCNT retained 92% of its starting activity under the same situations.At pH 8.0,free laccase preserved only 3% of its activity at 30 °C for 4 h,whereas the laccase immobilized on ZIF-8@MWCNT retained 23% of its initial activity at the same situations.When compared to free form,the relative activity of both enzymes was significantly reduced,but only slightly for laccase immobilized on ZIF-8@MWCNT,which could be directly linked to an increase in the number of hydroxyl ions in the close medium,limiting oxygen’s ability to bind to type 2 or type 3 laccase copper centers [44,48].Based on these findings,laccase immobilized on ZIF-8@MWCNT was more stable than the free form in all cases,owing to the formation of a strong attraction between the support and the laccase enzyme [39].

Thermal stability is the most significant factor in biocatalyst industrial applications [6].In this study,the thermal inactivation stability of the native enzyme was compared to that of the immobilized form.This comparison was performed under pH 3.0 and 65 °C after incubating immobilized and free laccase for various time intervals.As can be displayed in Fig.5(d),the free laccase retains 32% of its beginning activity at 65 °C for 180 min,whereas the immobilized laccase sustains 60% of its starting activity in the same situations.Laccase’s improved thermal stability following immobilization was attributed to the metal affinity capacity which renders a strong connection between ZIF-8@MWCNT support and laccase enzyme,which can decrease terrible conformational alteration at elevated temperature and thus protecting enzyme from denaturation [39,44,46].

3.4.Storage stability

After a few days of storage and reaction cycles,the immobilized enzyme might also sustain high activity and may also be deactivated under certain conditions when stored [48,50].In this study,the activity of free laccase reduced critically and 60%of the starting activity was maintained during the first 15 days and meanwhile only 42% of the original activity was maintained after 25 days(Fig.6).In comparison,the Laccase @ZIF-8@MWCNT still kept 86% and 77% of the original activity even after 15 and 25 days,respectively.The results displayed that ZIF-8@MWCNT support was tightly adsorbed the laccase enzymes to maintain the structural conformational changes,and enhanced the surrounding microenvironment to limit the denaturation of enzymes [13,44].Immobilized laccase activity may be reduced due to enzyme escape from the support material,but also microbial degradation and changes in the neighboring microenvironment [13,47,50].

Fig.6.Comparison storage stability of free and immobilized laccases at 4 °C for 25 days.

3.5.Reusability efficiency of immobilized laccase

The most critical key factor for industrial and environmental applications is the cost-efficient of the properties of enzyme reuse in multiple cycles [52].In this study,the reusability efficiency of the laccase immobilized on zeolitic imidazolate frameworks coated multi-walled carbon nanotubes (Laccase@ZIF-8@MWCNTs) was studied against 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)(ABTS)oxidation and dyes decolorization over multiple cycles in batch mode.The dyes decolorization and ABTS oxidation experiments were carried out as displayed in section:2.10 Reusability of immobilized laccase.Fig.7 clearly shows that laccase immobilized on ZIF-8@MWCNT could oxidize 10 batches of 1.2 mmol·L-1ABTS while keeping 68% remaining activity.

Fig.7.Reusability of the immobilized laccase using ABTS oxidation.

It was due to the properties of ZIF-8@MWCNT nanocomposite which assist the strong connection with the laccase enzyme,as well as metal affinity capacity which may cause a slight reduction in the laccase activity upon the reaction cycles[44].The reduction in activity after several cycles was ascribed to the leakage(escape)of the laccase upon use or utilization of low amount of biocatalytic system(10 mg,0.918 U) and enzyme inactivation or product inhibition[53,54].Similar findings were published where the immobilized laccase preserved 50% after ten cycles of oxidizing ABTS [55]and laccase immobilized on sepharose-linked antibody(44%)[54],which is lower than laccase immobilized on ZIF-8@MWCNT.The improved reusable of this synthesized biocatalytic system was due to the strong interaction between metal affinity adsorption on MWCNT with enzyme molecule [13,31].

Dyes decolorization by the immobilized laccase is a crucial factor for industrial and environmental applications [53-55].To implement this the immobilized laccase was utilized by decolorizing Acid Red 88 and Eriochrome Black T as model dyes and compared with that of the free laccase and ZIF-8@MWCNT.The results displayed that the ZIF-8@MWCNT immobilized with laccase (Laccase@ZIF-8@MWCNTs) had high decolorization efficiency for Eriochrome Black T (97.4%) and Acid Red 88 (95.6%) than its free form (92.72% and 91.7%,respectively) and individually ZIF-8@MWCNT (45.2% and 44.7%,respectively),and have been achieved in 25 min and the results were represented in Fig.8(a).The increased removal efficiencies for the ZIF-8@MWCNT immobilized with laccase contrasted to free laccase might be due to the close connection between the laccase immobilized onto ZIF-8@MWCNT and dyes,good compatibility of the substrate to the laccase molecule,and a high mass transfer rate [54,55].

Fig.8(b)displayed the decolorization efficiencies of Acid Red 88(AR 88)and Eriochrome Black T(EBT)dyes for ten sequential cycles of reutilization for 25 min each reaction.It was found that the decolorization percentages of AR 88 and EBT attained 96% and 98%for the immobilized laccase in the first cycle whereas individual ZIF-8@MWCNT achieved 50% and 59%,respectively.After ten batches,the decolorization efficiencies of AR 88 and EBT were approximately 73% and 78% for the immobilized laccase whereas individual ZIF-8@MWCNT reached 35% and 42%,respectively,proposing that the immobilized laccase was capable of the decolorization of azo dyes.The decrease in dye decolorization by immobilized laccase might be due to the inactivation or product inhibition of enzyme as well as enzyme escape during the batch operation [38,44,50,53].Despite the loss,the immobilized laccase indicated that the ZIF-8@MWCNTs nanocomposites could preserve the enzyme activity in practical use.

The results displayed that the immobilized laccase is capable of further decolorization of different dyes and hence more percentage color removal was attained employing immobilized laccase than free laccase and ZIF-8@MWCNT.However,to demonstrate the feasibility of this biocatalytic system,more experimental tests can be carried out at a higher dye concentration greater than 20 mg·L-1.Table 1 summarizes the decolorization procedures of dyes previously published,and as shown in the latter table,the developed method displayed high color removal in a shorter time without mediators compared to previously represented techniques,which showed a close connection between dyes and laccase immobilized on ZIF-8@MWCNT,and fast accessibility of laccase to the substrate[14,56],making laccase immobilized on ZIF-8@MWCNT more useful for industrial and environmental applications.

Table 1 A comparison of the color removal efficiency of the presented method and the previously reported works

Fig.8.(a) Decolorization kinetics and (b) decolorization reusabilities of azo dyes by the immobilized laccase and ZIF-8@MWCNT at 60 °C under pH 3.0.

4.Conclusions

The ZIF-8@MWCNT nanocomposites were successfully synthesizedviathe solvothermal method and employed for immobilizing laccase through the metal affinity adsorption process.pH 3 and 60 °C,respectively,were the pH and temperature optimum for immobilized laccase.The immobilized laccase displayed a broad pH range,improved pH,storage stability,and thermal stability in comparison to free laccase.Most notably,biocatalytic systems have been used to decolorize azo dyes and have shown great success compared to free enzyme.The potential use of ZIF-8@MWCNT immobilized laccase for dye decolorization indicates a promising future use in industrial or environmental applications.

CRediT Authorship Contribution Statement

Pascal Habimana:Methodology,Validation,Formal analysis,Visualization,Writing -original draft,Writing -review &editing.Yanjun Jiang:Methodology,Validation,Formal analysis,Visualization,Writing -original draft,Writing -review &editing,Supervision.Jing Gao:Formal analysis,Writing -review &editing,Supervision.Jean Bernard Ndayambaje:Writing -review &editing,Formal analysis.Osama M.Darwesh:Writing-review&editing.Jean Pierre Mwizerwa:Writing -review &editing.Xiaobing Zheng:Methodology,Validation,Formal analysis,Investigation,Writing-review &editing,Supervision.Li Ma:Methodology,Validation,Formal analysis,Investigation,Writing-review&editing,Supervision.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (Nos.21576068,21276060,21276062,and 21306039),the Natural Science Foundation of Tianjin City(16JCY-BJC19800),the Natural Science Foundation of Hebei Province (B2015202082,B2016202027,and B2020202036),the Science and Technology Program Project of Tianjin(20YDTPJC00260),the Program for Top 100 Innovative Talents in Colleges and Universities of Hebei Province (SLRC2017029) and Hebei High level personnel of support program (A2016002027).

Supplementary Material

Supplementary data to this article can be found online at https://doi.org/10.1016/j.cjche.2021.05.044.