Laccase immobilization on titania nanoparticles

Laccase immobilization on titania nanoparticles

and titania-functionalized membranes

Jingwei Hou,Guangxi Dong,Yun Ye,Vicki Chen n

UNESCO Centre for Membrane Science and Technology,School of Chemical Engineering,The University of New South Wales,Sydney,NSW2052,Australia

a r t i c l e i n f o

Article history:

Received17June2013

Received in revised form

2October2013

Accepted11October2013

Available online17October2013

Keywords:

Laccase immobilization

TiO2nanoparticles

Bio-catalytic membrane

a b s t r a c t

Bio-catalytic degradation of recalcitrant micropollutants with enzymes such as laccase provides an

environmentally attractive alternative to the conventional?ltration and adsorption processes.However,

enzyme loss and denaturation remain key challenges for their potential use in water treatment

applications.In this work,laccase immobilization on TiO2nanoparticles and TiO2blended polyethersul-

fone(PES)membranes were investigated due to TiO2's chemical stability,ease of functionalization,

and architecture.Different surface modi?cation and functionalization strategies on support materials

were compared based on enzyme loading,apparent activity,activity recovery,and stability.When

coupling agent3-aminopropyltriethoxysilane(APTES)and cross-linker glutaraldehyde(GLU)were

applied sequentially,effective coupling of laccase was achieved based on2,2′-azino-bis-(3-ethyl

benzothiazoline-6-sulfonic acid)(ABTS)assays.TiO2functionalized PES membrane showed better

enzyme immobilization ef?ciency than the non-functionalized membrane.Optimal performance was

observed for PES membrane containing4wt%TiO2,where TiO2not only provided the enzyme coupling

sites but also affected the membrane surface morphology and hydrophilicity to favor the enzyme

immobilization.These bio-catalytic membranes also displayed good enzyme stability,tolerance to wider

pH range and vigorous?ltration conditions required for water treatment applications.Kinetic study also

indicated that the enzyme af?nity to assay substrate was maintained after immobilization when

compared with packed bed and batch reactors.

&2013Elsevier B.V.All rights reserved.

1.Introduction

Interest in bio-catalytic systems has grown from both the

perspective of removal of recalcitrant compounds in water and

wastewater as well as energy generation in microbial and enzymatic

fuel cells.The presence of harmful micro-pollutant compounds,such

as pharmaceutically active chemicals(PhACs),hormone,pesticide and

endocrine disruptor chemicals(EDCs),in water source has become a

major health concern and efforts have been devoted to their removal

strategies[1–3].Laccase(polyphenoloxidase,E.C.1.10.3.2),a white-rot

fungi enzyme,can catalyze the oxidation of aromatic and related

compounds and shows good degradation ef?ciency for those pollu-

tants mentioned above[4,5].The use of laccase in micro-pollutant

elimination,xenobiotic degradation,and decolorization has been

extensively studied in the last decades[6–8],and it was reported to

be able to effectively remove the estrogenic activity of EDCs and

hormones[9].Therefore,the use of laccase for EDCs removal may

provide an alternative to conventional treatment methods,such as

ozone combined with UV,activated carbon adsorption,nano?ltration

and reverse osmosis.These conventional processes can generate

highly toxic by-products,have potential risk of adsorption and

leakage through membranes,and require signi?cant energy inputs

[8,10,11].Laccase has also been widely investigated as part of the

oxygen reduction reaction component of microbial and enzymatic

fuel cells[12].Recently Schaetzle et al.proposed laccase catalyst as an

enzymatic cathode reducing oxygen in combination with a microbial

fuel https://www.doczj.com/doc/b817185133.html,ccase was used in conjunction with ABTS redox mediator

to assist electron transfer between the cathode and the enzyme[13].

However,the issues associated with free enzymes such as instability

towards thermal and pH denaturation,inactivation by enzyme

inhibitors and poor reusability have impeded their application in

water treatment and fuel cells.To eliminate such shortcomings,the

enzyme immobilization has been proposed.

Enzyme immobilization can be achieved either by physical

adsorption or chemical immobilization(e.g.,covalent bonding and

cross-linking)[14].In physical adsorption,enzyme conformation is

largely preserved because the adsorption is mainly achieved by

either van der Waals'force or electrostatic interaction[15].

However,such bonding is relatively weak therefore causing enzyme

detachment from support during operation.In contrast,chemical

immobilization by covalent bonding or cross-linking usually provides

Contents lists available at ScienceDirect

journal homepage:https://www.doczj.com/doc/b817185133.html,/locate/memsci

Journal of Membrane Science

0376-7388/$-see front matter&2013Elsevier B.V.All rights reserved.

https://www.doczj.com/doc/b817185133.html,/10.1016/j.memsci.2013.10.019

n Correspondence to:School of Chemical Engineering,The University of New South

Wales,Sydney,2052,Australia.Tel.:t61293854813;fax:t61293855966.

E-mail address:v.chen@https://www.doczj.com/doc/b817185133.html,.au(V.Chen).

Journal of Membrane Science452(2014)229–240

much stronger bonding thus offering better stability and reusability compared to physical adsorption[6,16].For instance,3-aminopro-pyltriethoxysilane(APTES,silane coupling agent)is commonly used to provide aldehyde groups as anchor points.Subsequently,the cross-linking agent glutaraldehyde(GLU)is applied to bond with the amino groups on the protein surface.Unfortunately,these approaches normally lead to conformational changes of natural enzymes.As a result,lower speci?c activity of enzyme was com-monly observed[6,16].

Apart from different immobilization approaches,various sup-port materials with different chemical and nanostructure proper-ties have been explored.For instance,the immobilization of laccase on solid supports,such as soil[17],montmorillonite[17], CR633[18],mesoporous silica[19]and fumed silica nanoparticles [20]has been reported.A detailed discussion on different supports for laccase immobilization can be found in Duran et al.'s review [14].Characteristics of the support material(e.g.,size,surface property and morphology)have a signi?cant impact on immobi-lized laccase performance.Among different support materials, nanostructured materials have received increasing attention because:(i)they provide a higher surface area for enzyme attachment,and(ii)the high curvature of the nanoparticles, similar in size to proteins,allows a higher degree of freedom for the enzyme active sites and minimizes the lateral interaction between enzymes.Consequently higher enzyme activity was preserved[20–23].While nanoparticles provide high surface areas for immobilization,using functionalized microporous supports such as membranes avoids loss of the enzyme-particle complex through entrainment or adsorption.

Compared with other nanoparticles,the unique properties of TiO2nanoparticles,such as high mechanical strength,low price, physical and chemical stability,low toxicity,coordination ability with amine and carboxyl groups,as well as good biocompatibility, make it an ideal candidate for enzyme immobilization[24]. However,laccase immobilization on TiO2nanoparticles has sel-dom been reported in the literature.Nevertheless,the use of commercial TiO2nanoparticles as a support for other types of enzyme immobilization for biosensor preparation has been widely reported.Zhang et al.[25]prepared a biosensor by immobilizing lyophilized horseradish peroxidase onto unmodi?ed TiO2nano-particles.Better electro-catalytic activity was observed compared to the biosensors using nanoclay,chitosan,and gold nanoparticles as immobilization supports.Apart from the lyophilized horse-radish,the immobilization of hemoglobin[26],glucose oxidase [27],tyrosinase[28]and horseradish peroxidase[29]on commer-cial TiO2nanoparticles for biosensor preparation has also been explored.High activity and good stability were achieved after immobilization.Despite the good performance obtained from these works,it should be noted that only physical adsorption was applied in the enzyme immobilization in most studies,which could potentially lead to enzyme detachment over time as a result of the weak bonding.This is a critical issue particularly for more aggressive environments such as water and wastewater treatment processes.The presence of the hydroxyl groups on the TiO2surface provides a route for further chemical modi?cation of the particles' surface and further covalent enzyme-TiO2bonding which could potentially improve the immobilization performance.However, such modi?ed TiO2for covalent laccase immobilization has not been well studied to date.

While enzyme immobilization on nanoparticles can improve its stability,it still needs to be combined with other processes to enable the recycle and reuse of the particles and enzymes, especially in water treatment.In this line of development,the cascade?xed bed[30]and the perfusion basket reactor[31]were proposed previously to combine bio-catalytic nanoparticle and membrane?ltration together for wastewater treatment.Apart from such hybrid systems,bio-catalytic membrane,prepared by immobilizing enzyme on a micro-porous membrane support,may utilize the potential advantage of membrane separation process and provide higher mass transfer of the substrate to the enzyme. The application of bio-catalytic membrane reactor shows promis-ing application perspectives in food production,pharmaceutical industry and wastewater treatment process[32,33].For instance, after oxidization by laccase,bisphenol A(BPA)will form insoluble polymer which can be easily removed by the membrane?ltration process[34].Previous studies have been carried out on laccase immobilization on different membrane surfaces,such as poly-vinylidene?uoride(PVDF)membrane[4],polyamide membrane [35],polyethersulfone(PES)membrane[36]and chitosan mem-brane[37]to remove the micro-pollutants in an aqueous system.Covalent bonding is a common approach for immobiliz-ing enzymes on the surface of the polymeric membrane. The membrane surface is usually functionalized by GLU prior to enzyme immobilization.For example a thin gel layer was introduced onto the inorganic ceramic membrane initially, followed by GLU cross-linking and laccase immobilization. However,this membrane experienced severe fouling during ?ltration process[38].In order to improve the immobilization ef?ciency,dynamic adsorption(enzyme solution permeating through the membrane)was sometimes adopted instead of static adsorption by soaking in enzyme solutions[36,39]. However,those bio-catalytic membranes exhibited either low in activity recovery rate or poor in stability.In addition,the stability of immobilized laccase under membrane?ltration condition has not been extensively investigated.

Most previous works focused on the enzyme immobilization on either particles or membranes alone.Bio-catalytic nanoparticle is dif?cult to be used in wastewater treatment directly as recovery and reuse can be a major challenge,and the enzyme catalytic ef?ciency of the suspended nanoparticle might be reduced due to severe aggregation.On the other hand,the immobilization of enzyme on pure polymeric membranes was constrained by the drawbacks including the low activity recovery,poor stability and membrane fouling issues.So far few studies have been carried out regarding the enzyme immobilization on nanoparticle functiona-lized polymeric membranes which could potentially combine the bene?ts of the nanoparticle nanoparticles and membrane?ltration system.Blending TiO2nanoparticle into polymeric membranes to reduce the fouling propensity and increase the membrane poros-ity,or coating TiO2on the surface of the polymeric membrane to generate super-hydrophobic or super-hydrophilic surface has been extensively studied[40–44].Such a TiO2functionalized membrane for laccase immobilization can utilize the advantages of both the high activity recovery by TiO2nanoparticles and the convection of the substrate by membrane process.With this respect,the combined system with bio-catalytic particles and membrane ?ltration process could provide an alternative route for better immobilization performance.

In this study,bio-catalytic nanoparticles and membranes were prepared by the immobilization of laccase on TiO2and TiO2 functionalized PES membranes.Different laccase immobilization approaches including physical adsorption,sequential and GLU post-treatment were developed and examined in terms of laccase loading,activity,recovery rate and stability.TiO2functionalized PES membranes with different TiO2loadings were prepared to investigate the effect of TiO2loading on bio-catalytic membrane performance.In addition,the stability of bio-catalytic membrane under various?ltration conditions was investigated.Furthermore a kinetic study for both bio-catalytic nanoparticles and mem-branes was carried out and compared to provide a better under-standing of the effect of immobilization on the enzyme catalytic reaction.

J.Hou et al./Journal of Membrane Science452(2014)229–240 230

2.Experimental 2.1.Chemicals

Laccase from trametes versicolor (EC 1.10.3.2)(33U/mg)was supplied by Sigma.2,2'-Azino-bis-(3-ethyl benzothiazoline-6-sulfonic acid)(ABTS)from Sigma was used as the substrate for the laccase activity assay.TiO 2nanoparticles (Degussa,P25)were supplied by Degussa.3-Aminopropyltriethoxysilane (APTES)from Sigma and glutaraldehyde (GLU)from Ajax Finechem were used for TiO 2modi ?cation.Polyethersulfone (PES,molecular weight:58,000g/mol,BASF Co.Ltd.)was used to prepare the polymeric membrane.Polyvinylpyrrolidone (PVP,molecular weight:40,000g/mol)and dimethylacetamide (DMAc),both from Sigma,were used as membrane pore former and solvent respec-tively.All chemicals were of analytical grade and used without any puri ?cation.

2.2.TiO 2modi ?cation process

Before laccase immobilization,both physical and APTES mod-i ?cations of TiO 2were applied following the procedure previously reported by Razmjou et al.[43].For physical immobilization,the commercial TiO 2nanoparticles (Degussa P25)were ?rstly ground until its volume was reduced down to a quarter of its original level.The resultant powders were dispersed into pure ethanol and processed through both bath sonication (Transconic 46035kHz,Germany)and probe sonication (Misonic sonicators S-4000,USA,20amplitude)for 15min each.The resultant nanoparticles were referred as non-APTES modi ?ed TiO 2in this work.

After physical modi ?cation,the APTES modi ?cation was carried out by the following procedures:Diluted 10%(v:v)APTES in pure ethanol was added drop-wisely into TiO 2–ethanol suspension.Final mixture (total volume 50ml)contained 0.05g APTES and 2.5g of TiO 2.The reaction was conducted for 24h at 651C under nitrogen gas protection as the reaction was sensitive to water.Subsequently,these nanoparticles were separated by centrifuga-tion and redispersed in Milli-Q water for 3times to wash off all the unreacted APTES.The ?nal nanoparticles were dried at 551C and then ground into ?ne powder again.The resultant nanoparticles were referred as APTES modi ?ed TiO 2in this work.2.3.Preparation of bio-catalytic TiO 2nanoparticles

To investigate the effect of immobilization procedure on the bio-catalytic nanoparticle performance,3different approaches for laccase immobilization were applied in this work.

Physical adsorption :Non-APTES modi ?ed TiO 2and APTES modi ?ed TiO 2were both used directly for physical adsorption.Fifty miligram of TiO 2was suspended in 5ml laccase solution (100m g/ml)for 60h at 41C.Unreacted laccase was removed by

centrifugation and re-dispersion in Milli-Q water for 3times until no laccase activity was detected in the supernatant.

Sequential immobilization :In this process 50mg of APTES modi ?ed TiO 2was dispersed in 4%(v:v)GLU in pH 7S?rensen's phosphate buffer solution for 12h to allow the functionalization,then all the unreacted GLU was washed off by centrifugation and re-dispensation for 3times in S?rensen's phosphate buffer.The GLU functionalized TiO 2particles were suspended in 5ml of laccase solution (100m g/ml)for 60h at 41C.After that,the unreacted laccase was removed following the same procedure as described in the physical adsorption (as shown in Fig.1a).

GLU post-treatment :Unlike the sequential immobilization,APTES modi ?ed TiO 2was suspended in laccase solution (100m g/ml)for 60h at 41C without the GLU functionalization step,then the nanoparticles were separated and resuspended in 4%(v:v)GLU solution for 12h at 251C.Finally the unreacted GLU was removed by centrifugation and re-dispensation (as shown in Fig.1b).

All bio-catalytic nanoparticles were suspended in Milli-Q water and stored at 41C for future testing.2.4.Preparation of bio-catalytic membranes

Membranes were prepared by a phase inversion technique similar to the process reported by Razmjou et al.[43].The casting solutions contained 16wt%PES,4wt%PVP as the pore former,and DMAc as the solvent.Different amounts (0,1,2,4and 6wt%in casting solution)of TiO 2were added to evaluate the effect of TiO 2loading on the laccase immobilization performance.After degas-sing overnight,the casting machine (Sheen 1133N automatic ?lm applicator)was used to cast the membranes under 50%humidity and 60mm casting speed.The nascent membranes were allowed to coagulate in the Milli-Q water bath for 24h.The membrane was further rinsed with 100ml Milli-Q water for three times to remove the loosely attached TiO 2.The stability of attached TiO 2was examined by ?ltering Milli-Q water through membrane at 400L/m 2h for 5h.The thermogravimetric analysis (TGA)showed no loss of TiO 2content after ?ltration,indicating good stability of the TiO 2in the membrane.

Similar to laccase immobilization on TiO 2nanoparticles,3differ-ent approaches were used for laccase-membrane immobilization.

Physical adsorption :Commercial PES membrane (10kDa,from Millipore),in-house pure PES membrane and in-house PES mem-brane containing 4wt%non-APTES modi ?ed TiO 2(referred as PES-4%TiO 2)were applied for laccase physical adsorption as benchmarks.Enzyme immobilization was accomplished by soak-ing 2cm 2membranes into 5ml laccase solution (100m g/ml)for 60h at 41C.

Sequential immobilization :The APTES-modi ?ed TiO 2was functionalized with GLU before being added into the PES casting solution for membranes https://www.doczj.com/doc/b817185133.html,ccase was immobilized on this series of membranes by the same procedure described

above.

Fig.1.Schematic representation of different protocols to immobilize laccase on TiO 2nanoparticles.(a)Sequential process and (b)GLU post treatment.

J.Hou et al./Journal of Membrane Science 452(2014)229–240231

For different TiO2loadings,this series of bio-catalytic membranes were referred as PES-1%TiO2-Seq,PES-2%TiO2-Seq,PES4%TiO2-Seq,and PES-6%TiO2-Seq(containing1,2,4and6wt%TiO2in the casting solution respectively).

GLU post-treatment:In this series of membranes,APTES modi?ed TiO2was used to prepare the membranes.After immer-sing the membranes in laccase solution for60h at41C,these membranes were transferred to4%(v:v)GLU solution to allow the cross-linking to take place at251C for12h.This series of bio-catalytic membranes were referred as PES-1%TiO2-GLU-post,PES-2%TiO2-GLU-post,PES4%TiO2-GLU-post,and PES-6%TiO2-GLU-post depending on the TiO2loadings.

2.5.Assessment of laccase activity

Laccase activity can be analyzed using different substrates including ABTS,2,4-dichlorophenol(2,4-DCP),2,6-dimethoxyphe-nol(DMP),dihydroxyphenylalanine(DOPA),dimethylaminobor-ane(DMAB)and syringaldazine.Each substrate has different af?nity to free and immobilized laccase.ABTS was applied in most recent studies as it is3times more sensitive than phenol based substrates(e.g.DMP)and over40times more sensitive than other assay substrates(e.g.DOPA,DMAB,and syringaldazine)[45].

Laccase activity was determined by monitoring the oxidation rate of ABTS to ABTStat420nm using a Cary100UV–visible Spectrophotometer[46].Each test was conducted in a total volume of20ml at251C.For free enzyme,0.5ml laccase solution was added into19.5ml ABTS solution(0.5mM ABTS in pH3 phosphate–citrate buffer solution).For bio-catalytic particles, 50mg of laccase functionalized TiO2was dispersed in20ml ABTS solution.Before the UV measurements,the solution was rapidly ?ltered through a Millipore0.1m m syringe?lter to remove the bio-catalytic particles.For the bio-catalytic membrane,membranes with2cm2in size were suspended in20ml ABTS solution.The absorbance was measured once per minute.One unit(U)of laccase activity was de?ned as the amount of laccase forming1m mol of ABTStper minute.

The activity recovery was de?ned as the apparent activity of the immobilized laccase divided by the laccase activity theoreti-cally immobilized onto the support.The theoretically immobilized activity was calculated as the activity difference in laccase solution between the initial laccase activity before immobilization and the residual activity at the end of the immobilization procedures after the washing step.The binding yield was de?ned as the ratio between the activity theoretically bond to the support and the total initial laccase activity in solution before immobilization.A more rigorous term“effective binding yield”was de?ned to better re?ect the ef?ciency of immobilization,which was the ratio between the apparent activity of the immobilized laccase and the initial laccase activity in solution before immobilization.

2.6.Stability of the immobilized enzyme

The stability of immobilized laccase at room temperature was investigated by measuring the residual activity of bio-catalytic TiO2nanoparticles and bio-catalytic membranes daily for up to20 days,and results were compared with the initial activity.The stability of immobilized laccase under chemical exposure,different pH and?ltration conditions was also examined in this study.

2.7.Determination of kinetic parameters

The Michaelis–Menten kinetic parameters K m and k cat of free laccase,bio-catalytic nanoparticles and bio-catalytic membranes were determined by measuring the laccase activity using ABTS as substrate under different substrate concentrations ranging from 5to2000m M.The values of the kinetic parameters were obtained by non-linear curve?tting of the plot of reaction rate versus substrate concentration based on the following equation:

ν?V max?S

mt?S

?k cat E? 0

?S

mt?S

e1T

1

V

?K m

V max?S

t1

V max

e2T

where v is the reaction rate,and[S]is the substrate concentration, V max represents the maximum rate achieved by the bio-catalytic system.The Michaelis constant K m is the substrate concentration at which the reaction rate is half of V max and is often used as the indication of the enzyme's af?nity to the substrate.[E]0is the enzyme concentration.k cat(m mol sà1gà1)is the maximum num-ber of substrate molecules converted to product per enzyme molecule per second.k cat K mà1is a measurement of the ef?ciency of enzyme to convert the substrate into product.

2.8.Membrane characterization

Membrane structure and distribution of TiO2particles within the membrane was investigate by FEEM-EDS(Hitachi S3400).The cross section of the membrane samples were prepared by fractur-ing the membrane in liquid nitrogen and coated with chromium [43].

In terms of membrane?ltration performance,a dead-end cell ?ltration system was set up to measure the Milli-Q water?ux of the membranes.The details of the?ltration system can be found elsewhere[43].The effective membrane area was0.0014m2.

The molecular weight cut-off(MWCO)of the membranes was measured by monitoring the rejection of dextrans with different molecular weight(26,70,260and476kDa,respectively)[43].The dextran concentrations in both the feed and the permeate were analyzed by total organic carbon analyzer V-CSH(Shimadzu TOC-VCSH&Auto-sampler ASI-V).

3.Results and discussion

3.1.Performance of the bio-catalytic nanoparticles

3.1.1.Effect of immobilization protocols

Laccase was immobilized onto different TiO2nanoparticles via various immobilization approaches and the results along with the literature values are summarized in Table1.In terms of the physical adsorption,higher apparent activity was observed on the APTES modi?ed TiO2(0.08570.011U/mg support)in compar-ison with the non-APTES modi?ed TiO2(0.07070.020U/mg sup-port)while their laccase loadings were similar.Previous work from our group showed that the average particle size of non-APTES modi?ed TiO2was160nm with a polydispersity of0.26, while the particle size of APTES modi?ed TiO2reduced to84.2nm with narrower polydispersity of0.19[43].The amount of laccase immobilized on TiO2did not change with the decrease of TiO2 particle size in this work.However higher apparent activity and activity recovery were observed for smaller sized TiO2.By contrast, the increase in enzyme loading while reducing the particle size was reported previously by Jia et al.onα-Chymotrypsin immobi-lization on polystyrene particles(ranging from110to1000nm) [47].In their study,higher apparent activity was also observed for smaller size nanoparticles.The slightly higher mobility of smaller sized enzyme-immobilized particle might result in higher appar-ent activity of immobilized enzymes[47].It was consistent with the effect of particle size on enzyme-catalytic reaction based on the Collision theory[48].

J.Hou et al./Journal of Membrane Science452(2014)229–240 232

In terms of the effect of immobilization protocols on the immobilization performance,sequential technique exhibited the highest apparent activity (0.13570.015U/mg support)and activ-ity recovery (7976%)with the lowest loading (5.170.5m g/mg support),while the GLU post-treated particles showed the highest laccase loading (8.671.0m g/mg support)but the lowest activity (0.07070.013U/mg support)and activity recovery (2475%).In comparison,the physically immobilized laccase exhibited mediate activity recovery for both sized supports (4075and 4876%).Physical adsorption process is normally expected to have high activity recovery as the laccase's natural conformation could be well preserved.However,in this work high speed centrifugation process was applied in the bio-catalytic nanoparticle preparation to remove unreacted enzyme.High shear and pressure forces between laccase-laccase and laccase-support during the centrifuge could potentially change enzyme's natural conformation,reducing the immobilized enzyme's apparent activity and activity recovery.In comparison,for covalent bonded laccase,the cross linker between laccase and support provided higher enzyme rigidi ?ca-tion therefore enzyme's activity was less sensitive to further centrifugation process.By using GLU as a covalent bonding agent for the post-treatment,strong chemical linkage could be formed not only between the laccase and support but also between the laccase molecules,thus forming a stable network structure,which increased the laccase loading on the nanoparticle surface.However,this structure could change laccase conformation and reduce the af ?nity between the laccase active site and substrate.As a result,a much lower activity recovery was observed.In comparison,during the sequential process,the covalent linkages were mainly formed between TiO 2and laccase,not between laccase molecules.Such a structure provided higher mobility of the immobilized laccase.As a result,higher apparent activity and activity recovery rate were observed in the sequential process despite of the moderate laccase loading.

Similar results were also reported by Cabana et al.where laccase was immobilized on CR633with different immobilization procedures [18].In their study,it was found that the sequential immobilization had much higher apparent activity even with lower binding ef ?ciency,resulting in an activity recovery of 5578%.On the contrary,much lower apparent activity even with 100%binding ef ?ciency was observed in the simultaneous GLU immobilization process where GLU was added into the laccase solution during immobilization.Similar to GLU post-treatment process in the current work,a strong cross-linking network was formed and led to lower activity recovery (Table 1).A compre-hensive investigation was carried out by Zimmermann et al.[20]regarding the effects of different immobilization procedures on the enzyme catalytic performance.In their study,improved binding ef ?ciency through cross-linking of laccase with GLU was also observed.However,unlike what was observed in our and Cabana's works,higher apparent activity was reported using GLU cross-linking approach than the sequential process.Such a difference might be due to the different GLU concentration applied.In Zimmermann's work [20],much higher apparent activity was observed in the GLU cross-linking procedure when excessive amount of GLU was used.Previous work has suggested that GLU can be considered as a stabilizer as it can promote the multipoint attachments and enzyme rigidi ?cation,thus reducing the unfold propensity of the enzymes.Better protein thermo-stability was observed from Demarche's work when increasing the GLU con-centration [19].

Much higher apparent activity (1.5–2.7U/mg fumed silica nanoparticle)was reported in Zimmermann's work regardless of the immobilization procedure used (Table 1)[20]in comparison with our work.Such results might be attributed to:(i)their nanoparticles were exposed to a much higher initial enzyme

T a b l e 1P e r f o r m a n c e o f i m m o b i l i z e d l a c c a s e o n T i O 2n a n o p a r t i c l e s u s i n g d i f f e r e n t a p p r o a c h e s .

I m m o b i l i z a t i o n p r o c e d u r e S u p p o r t

A p p a r e n t a c t i v i t y (U /m g s u p p o r t )L a c c a s e L o a d i n g (m g /m g s u p p o r t )

T h e o r e t i c a l a c t i v i t y (U /m g s u p p o r t )B i n d i n g e f ?c i e n c y (%)A c t i v i t y r e c o v e r y (%)E f f e c t i v e b i n d i n g y i e l d (%)R e f e r e n c e

P h y s i c a l a d s o r p t i o n N o n -A P T E S m o d i ?e d T i O 2

0.07070.0205.370.50.1870.02537540752173C u r r e n t w o r k P h y s i c a l a d s o r p t i o n A P T E S m o d i ?e d T i O 2

0.08570.0115.370.60.1870.03537648762574C u r r e n t w o r k S e q u e n t i a l A P T E S m o d i ?e d T i O 2

0.13570.0155.170.50.17170.019517579764074C u r r e n t w o r k G L U p o s t t r e a t m e n t A P T E S m o d i ?e d T i O 2

0.07070.0138.671.00.2970.048671024752073C u r r e n t w o r k

S e q u e n t i a l C R 6330.008–0.01455784579–[18]G L U s i m u l t a n e o u s C R 6330.003–0.021001571–[18]P h y s i c a l a d s o r p t i o n F u m e d s i l i c a n a n o p a r t i c l e s 1.5––32.860–[20]S e q u e n t i a l F u m e d s i l i c a n a n o p a r t i c l e s 1.47––29.865–[20]G L U s i m u l t a n e o u s F u m e d s i l i c a n a n o p a r t i c l e s 2.67––61.457.9–[20]

J.Hou et al./Journal of Membrane Science 452(2014)229–240233

amount during the immobilization process (3.0–7.5U laccase/mg support)than that in our study (0.33U laccase/mg support).A higher initial enzyme amount ensures more enzyme adsorbed onto the support surface,which may result in higher enzyme loadings and higher apparent activity,and (ii)particles with much higher speci ?c surface area (SSA)(320.3716.1m 2/g for fumed silica nanoparticle)were used in their work in comparison with the SSA of the TiO 2particles in this study (60.170.3m 2/g).In their study,immobilization using spherical nanoparticles with surface area of 21m 2/g also resulted in lower apparent activity (0.2370.01U/mg support,less than 10%of apparent activity obtained from fumed silica nanoparticle).Therefore,the initial laccase concentration applied in immobilization could be further optimized to improve the performance of the bio-catalytic nano-particle in this work.

3.1.2.Stability of the laccase immobilized on particles

The stability of laccase immobilized on TiO 2particles was assessed and the results are shown in Fig.2.The immobilized laccase were suspended in 0.1M phosphate buffer (pH ?7)at room temperature (251C)for a period of 20days.For the laccase physically adsorbed on the support,drastic drop of activity (nearly 50%)was observed in the ?rst 6days,followed by a slower decrease during the remainder of the period.Much better stability was observed for the laccase immobilized via both sequential immobilization procedure and GLU post-treatment procedure,where over 90%activity was maintained.In the case of physical adsorption,the loose attachment of laccase by van der Waals'force cannot provide stable bonding for enzymes.Covalent immobiliza-tion (e.g.sequential or GLU post-treatment),on the other hand,offered strong chemical bonding between laccase and support,and the covalent linkage formed between laccase and TiO 2improves the structural rigidi ?cation of laccase,thus leading to a better stability.Even though no obvious difference was observed between sequential and GLU post-treatment processes in terms of the stability,the sequential process still exhibited higher remaining activity (0.12U/mg support)than GLU post treatment process (0.06U/mg support)at the end of the stability test due to its higher initial activity value.As a result,the sequential process

was regarded as the optimized immobilization approach for nanoparticles.

3.1.3.Kinetics of the immobilized laccase on TiO 2particles

Table 2shows the Michaelis –Menten kinetic constants for the oxidation reaction of ABTS catalyzed by laccase immobilized on TiO 2using different immobilization procedures.The K m for free laccase was 22.372.0m M.After immobilization,the K m values for laccase increased 2.5–4.5times,indicating the loss of af ?nity of enzyme to substrate.The increase of K m after immobilization could be attributed to the internal and external limitations,such as mass transfer resistance and limited accessibility of active sites after immobilization.The conformational changes of the protein molecule and steric hindrance could also lead to an increase in K m .Among three different immobilization procedures,the sequential immobilization procedure exhibited the least loss in af ?nity,which was 2.5times lower than the free laccase.By contrast,the af ?nity of laccase immobilized by the GLU post treatment and the physical adsorption procedure were 4.5and 4.2times lower than that of the native laccase.The increase in K m is commonly observed on enzyme immobilization studies.In comparison,Cabana et al.[18]reported a 6times increase in K m when immobilizing enzymes on CR633via the GLU sequential immobilization procedure,and a 20times increase when applying the simultaneous GLU treatment procedure.Cabana's work indicated that the GLU sequential process offered the lowest laccase af ?nity to substrate which was in agreement with our work.

As indicated in Table 2,k cat /K m decreased after immobilization indicating the loss in laccase ef ?ciency to convert substrate into product.The sequential immobilization process showed the high-est ef ?ciency among all three immobilized approaches.Similar kinetics study on immobilized laccase was performed by Cabana et al.[18].In their study,k cat /K m after immobilization on CR633reduced to less than 1%of the free laccase.Above results also indicated that,by using TiO 2nanoparticles as immobilization support,the ef ?ciency of immobilized laccase was better pre-served than CR633.

3.2.Performance of the bio-catalytic membrane

3.2.1.Effect of immobilization protocols

The effect of immobilization procedures on bio-catalytic mem-brane performance is showed in Fig.3.In terms of laccase loading,physical adsorption process showed higher loading than covalent process,and the highest laccase loading was observed on PES-4%TiO https://www.doczj.com/doc/b817185133.html,pared with PES in-house membrane,PES-4%TiO 2membrane only exhibited slightly loading improvement.After blending unmodi ?ed TiO 2into PES membrane matrix,there

R e s i d u a l A c t i v i t y (%)

Time (day)

Fig.2.Activity pro ?les of immobilized laccase on TiO 2nanoparticles as a function of time (stored at 251C).

Table 2

Kinetic parameters of the enzyme immobilized on TiO 2nanoparticles.

Free laccase

Physical

Sequential

GLU-Post treatment k cat (m mol s à1g à1)496721236725382732151718K m (m M)

22.372.092.7710.756.073.0100.8713.4k cat K m à1(L s à1g à1)

22.371.8 2.5570.24 6.8270.53

1.5070.20

0.000.040.080.12

0.16

A c t i v i t y (U /c m 2)

35

70

105

140

L a c c a s e L o a d i n g (μg /c m 2 m e m b r a n e )

Fig.3.Bio-catalytic membrane activity (■)and laccase loading ()by different

immobilization techniques.

J.Hou et al./Journal of Membrane Science 452(2014)229–240

234

would be TiO 2exposed on the membrane surface,which provided extra anchor positions for laccase physical adsorption.However,the marginal loading improvement for PES-4%TiO 2membrane indicated most laccase was still adsorbed on PES polymer surface in this bio-catalytic membrane system.In comparison,the incor-poration of APTES modi ?ed TiO 2particles in the polymeric membrane resulted in a substantial reduction in surface roughness [43],leading to lower surface area and further lower laccase loading for covalent procedures.

In terms of the bio-catalytic membrane activity,it is unex-pected that the covalently bonded laccase exhibited higher activity than the physical adsorbed laccase despite of the lower laccase loading (Fig.3).In the case of laccase physically adsorbed on the membrane surface,most of the laccase was immobilized on the bulk hydrophobic PES polymers.The hydrophobic core of laccase could be oriented towards to the polymer surface leading to laccase denaturation [43,49].Whereas in the case of the covalently bonded laccase,the negative impact from bulk hydrophobic PES polymer was off-set by the strong chemical bonding between laccase and TiO 2particles.More speci ?cally,in the case of the sequential process,the formation of the single linkage rigidi ?ed the laccase structure while still allowed good laccase mobility,therefore the highest activity was observed from this process.In comparison,in the GLU post-treatment process,the formation

of the network structure restrained the laccase mobility which weakened the positive impact from the rigidi ?cation of laccase.Therefore a substantial loss of activity was observed in comparison with the sequential process.In addition,the laccase was immobi-lized via two different modes in the sequential and the GLU post-treatment processes:(i)covalent bonding between TiO 2and lac-case,and (ii)physical adsorption between TiO 2-laccase and PES polymer-laccase.However in the case of mode (ii),the physical adsorption between particles and laccase was minimal,as the low af ?nity between the hydrophilic TiO 2nanoparticles and the hydrophobic laccase led to a negligible laccase loading.Further-more,the strong interaction between the hydrophobic PES poly-mer and the hydrophobic laccase reduced the immobilized laccase activity.With these regards,mode (i)dominated the immobiliza-tion mechanism in the sequential and GLU post-treatment processes.

Fig.4compares the molecular weight cut-off (MWCO)of three bio-catalytic membranes and no signi ?cant pore size difference was https://www.doczj.com/doc/b817185133.html,ccase used in this study has a molecule weight around 70kDa,which is lower than the MWCO of all the membranes used in this work,indicating that laccase could potentially penetrate into the membrane matrix and attach inside the membrane pores apart from being immobilized only on the membrane surface.Higher permeate ?ux was observed from the PES-4%TiO 2-Seq membrane than the in-house PES membrane before laccase immobilization (Table 3)indicating slightly larger pores or higher porosity for the PES-4%TiO 2-Seq membrane,which was consistent with the molecular weight cut-off results (Fig.4).Table 4shows a more signi ?cant ?ux drop on the PES-4%TiO 2-Seq membrane after laccase immobilization even though it had lower laccase loading than the in-house PES membrane.This suggested that,for the PES-4%TiO 2-Seq membrane,most laccase was located inside the membrane pores rather than on the membrane surface.The results indicated that different immobili-zation procedures not only in ?uenced the laccase loading but also the position where laccase was anchored,which further affected the binding ef ?ciency and activity recovery.

The performances of all the TiO 2functionalized bio-catalytic membranes are summarized in Table 4.In this work,the activity recovery for the bio-catalytic membranes was found to be much lower than those for the bio-catalytic TiO https://www.doczj.com/doc/b817185133.html,ccase immobilized on the membrane could be attached on both polymer matrix and TiO 2particles,but only the laccase attached on the particles was covalently bonded due to the existence of functional group (aldehyde group)on the particle surface after APTES modi ?cation.However,as some of the TiO 2nanoparticles were buried inside the membrane polymer matrix and poorly accessible by laccase to be functioned as the immobilization support,the activity recovery was therefore expected to be lower for bio-catalytic membranes than particles.Furthermore,the lower mobility of the laccase on the membrane than on TiO 2led to a higher mass transfer

020406080

100R e j e c t i o n (%)

MW of Dextran (kDa)

Fig.4.Molecular weight cut-off for different membrane systems.

Table 3

Milli-Q water ?ltration test results before and after laccase immobilization for different types of membranes (trans-membrane pressure:1bar,membrane area:0.0014m 2).

Before laccase

immobilization (L/m 2h)

After laccase

immobilization (L/m 2h)In-house PES

membrane 300730220715PES-4%TiO 2-Seq

350715

170712

Table 4

Performance of in-house fabricated TiO 2functionalized bio-catalytic membranes and other bio-catalytic membranes from literature.

Membrane

Activity (U/cm 2membrane)Laccase loading (m g/cm 2membrane)Activity

recovery (%)Reference

Physical adsorption

In house PES 0.04470.008109710 1.270.1Current work PES-4%TiO 2

0.05870.006123713 1.470.2Current work GLU post treatment PES-4%TiO 2-GLU-Post 0.06170.0089576 1.970.3Current work Sequential

PES-4%TiO 2-Seq 0.13170.01352757.470.4Current work

Physical adsorption

Poly HEMA ?lm 0.099139 3.55[49]Glutaraldehyde cross linking Ceramic membrane –

147–[38]Glutaraldehyde cross linking

Polyamide 6,6fabric

0.085–10[50]Physical adsorption (enzyme ?ltrate

through membrane)

Commercial PES membrane (3000Da)

Up to 9.8

–

425

[36]

J.Hou et al./Journal of Membrane Science 452(2014)229–240235

resistance which further contributed to the lower activity recovery,such a behavior was also reported by Jia et al.[47].

Compared with values in literature (as shown in Table 4)[38,50,51],poly HEMA membranes and ceramic membranes had comparable performance to our bio-catalytic membranes,only polyamide 6,6fabric had higher activity recovery which might be due to the larger surface area offered by the fabric system.A much higher activity (up to 9.8U/cm 2membrane)and activity recovery (425%)was reported by Lante et al.[36]on the PES membrane with a MWCO of 3kDa.This better performance could be attrib-uted to the different immobilization approach applied (dynamic adsorption,?ltering laccase solution through the membrane).This approach might cause the formation of a thick laccase cake layer on the membrane surface resulting in much higher activity and activity recovery,considering the much larger laccase molecules (70kDa)than the pore size of the membrane (MWCO:3kDa).However,this laccase cake could also increase the membrane

resistance thus lowering the membrane ?ltration performance.It should also be noted that such an enzyme cake with closely packed laccase molecules might pose signi ?cant in ?uence on the stability of bio-catalytic membrane.However this issue was not addressed in their work.

3.2.2.Effect of TiO 2concentration on functionalized membranes As shown in Fig.5,the optimum TiO 2loading was appeared at 4wt%where the best apparent activity and activity recovery were achieved for both sequential and GLU post-treatment procedures.Higher TiO 2loading offered more available anchor positions on the particles surface which resulted in higher laccase loading and apparent activity.Apart from TiO 2loading,a good distribution of TiO 2nanoparticles without aggregation is another key issue.Further increasing the TiO 2loading to 6wt%led to the particle aggregation (as shown in Fig.6).Similar aggregation of TiO 2at high concentration was also observed by Razmjou et al.[43].In addition,the large aggregates may not adhere well with the polymer matrix thus could be easily leached into the coagulation medium during the membrane preparation process,limiting the actual particle loading in the resultant membrane [40].It should be noted that the Ti signals (Fig.6C and D)observed above the membrane cross-section image were in fact the Ti particles present on the membrane upper surface.As the sample was not placed perfectly upright during the EDX analyze,the surface signals were also collected.This observation also proved that even though in the blended membrane system TiO 2nanoparticles were buried within the PES polymer matrix,there was still considerable number of TiO 2nanoparticles exposed on the membrane surface,readily for laccase immobilization.

The average Milli-Q water ?uxes for the bio-catalytic mem-branes measured under the feed pressure of 1bar also con ?rmed the effect of TiO 2loading on the activity and recovery rate of the

0.000.040.080.12

0.16A c t i v i t y R e c o v e r y (%)

A c t i v i t y (U )

2

4

6

8

Fig. 5.Apparent activity (■)and activity recovery ()of the bio-catalytic

membranes with different TiO 2loadings.

Fig.6.SEM and EDX images of the bio-catalytic membranes with different TiO 2loadings.(a)PES-4%TiO 2-Seq membrane,(b)PES-6%TiO 2-Seq membrane,(c)EDX image of PES-4%TiO 2-Seq membrane and (d)EDX image of PES-6%TiO 2-Seq membrane.

J.Hou et al./Journal of Membrane Science 452(2014)229–240

236

bio-catalytic membranes (Fig.7).Higher Milli-Q water ?ux was observed on the membranes with 4wt%TiO 2loading indicating larger membrane pores and porosity which in return offered lower mass transfer resistance between substrate and immobilized lac-case,which resulted in higher apparent activity.Further increase of the particle loading to up to 6wt%led to a drop in Milli-Q water ?ux which might be partially attributed to the aggregation of the particles [43].The existence of an optimum TiO 2loading on water ?ux was also observed by Razmjou et al.[43].TiO 2loading of 2wt %was reported as the optimum concentration in their study instead of 4wt%observed from this work.This might be due to the different TiO 2modi ?cation techniques adopted between two studies,where in the current study TiO 2was further treated with GLU for enzyme immobilization.

3.2.3.Stability of the bio-catalytic membrane

Fig.8shows the activity pro ?le of the bio-catalytic membranes stored at room temperature (251C)during a period of 20days.The pure in-house PES membrane and the PES-4%TiO 2membrane lost nearly 80%of their original enzyme activity within the ?rst 5days caused by the loose physical attachment between the enzymes and supports.For the covalently prepared bio-catalytic mem-branes,improved stabilities were observed:75%activity was preserved for the PES-4%TiO 2-GLU-Post membrane and 40%for the PES-4%TiO 2-Seq https://www.doczj.com/doc/b817185133.html,pared with the single-point bonding formed by the sequential process,the multi-point cross-linking caused by the GLU post-treatment provided higher enzyme rigidi ?cation and better preserved laccase's natural conformation over time.However it was noted that the PES-4%TiO 2-Seq membrane still maintained higher activity than that of the PES-4%TiO 2-GLU-Post membrane at the end of the testing period.

With this regard,the sequential process was still considered as the preferable immobilization approach in this study.In comparison with the bio-catalytic TiO 2particles (Fig.2),bio-catalytic mem-branes showed lower stability which might be due to the sub-stantial fraction of the laccase was loosely attached on the polymer matrix rather than covalently bonded on the TiO 2particles.

The effect of TiO 2loading on the membrane stability was also assessed.Fig.9demonstrates that the TiO 2concentration has very little in ?uence on the bio-catalytic membrane stability.This observation suggested that the stability of the bio-catalytic mem-branes were mainly determined by the immobilization procedure.Even though the GLU post-treatment exhibited better stability,due to higher initial activity,the sequential process still showed higher absolute activity after 20days (0.040U cm à1for PES-2%TiO 2-Seq compared with 0.028U cm à1for PES-2%TiO 2-GLU-Post).In our study the stability tests were conducted for 20days as the remaining activity pro ?les were considerably stabilized by the end of this period.Stability tests with longer periods will be carried out in our future study,to better demonstrate the feasi-bility of utilizing such a bio-catalytic system in a real industrial environment.

The stability of the bio-catalytic membranes under ?ltration conditions at the ?ux of 400L/m 2h was also examined (Fig.10).The tendencies were consistent with the stability of the mem-branes stored at 251C.Pure PES membrane lost nearly 90%of initial activity after ?ltering 800ml of Milli-Q water through the membrane.By contrast,better stability was observed on the PES4%TiO 2-GLU-Post membrane:Fifty ?ve percent of activity maintained after ?ltering 10L of Milli-Q water.For the PES4%TiO 2-Seq membrane,30%activity was lost during initial 500ml and the activity loss then followed a similar pattern as the PES-4%TiO 2-GLU-Post membrane.It should be noted that ?ltration

A c t i v i t y (U /c m 2)

Time (day)

Fig.8.Stability of the bio-catalytic membranes with different immobilization techniques at room temperature (251C).

R e s i d u a l a c t i v i t y (%)

Time (day)

Fig.9.Stability of the bio-catalytic membranes with different TiO 2loadings at room temperature (25o C).

A c t i v i t y (U /c m 2)

Total Process Volume (L)

Fig.10.Stability of the bio-catalytic membrane under vigorous Milli-Q water ?ltration (constant ?ux at 400L/m 2h with dead-end

set-up).

80160240320

400

F l u x (L /m 2h )

Fig.7.Pure water ?ux for different bio-catalytic membranes with trans-membrane pressure of 1bar.

J.Hou et al./Journal of Membrane Science 452(2014)229–240237

condition used in this study (400L/m 2h)was much vigorous than the permeate ?ux normally applied in practical water https://www.doczj.com/doc/b817185133.html,li-Q water ?ltration test under modest condition (10L/m 2h)was also performed with all bio-catalytic membranes.Results also proved the stability of enzyme immobilized membranes with covalent bonding were better:the activity of the PES-4%TiO 2-GLU-Post and the PES-4%TiO 2-Seq membranes only reduced by 10%and 15%respectively after ?ltrating up to 10L of Milli-Q water under 10L/m 2h.

To examine the chemical resistance of the bio-catalytic mem-branes,membranes were exposed to different denaturants for

different period of time.The membranes were soaked in three chemical solutions:0.1wt%NaOH,25%(v:v)MeOH and 25%(v:v)acetone for 5min,1min and 1min respectively.The pure in-house PES membrane lost most of its activity after incubation regardless the types of denaturants,while the PES-4%TiO 2-GLU-Post mem-brane exhibited the best chemical resistance among all three membranes (Fig.11).Without the aid of GLU cross-linking,in-house PES and PES-4%TiO 2-Seq membranes lost their activities rapidly after exposure to 1wt%NaOH solution for 5min.NaOH is often used as membrane chemical cleaning agent to remove proteins from the membrane surface.The results from this work suggest that NaOH is highly detrimental for bio-catalytic mem-brane containing laccase.Reloading of enzyme would probably be necessary after such a chemical exposure.

The effect of pH on the bio-catalytic membranes was also studied by incubating the free laccase and immobilized laccase in buffer solutions with different pH values ranging from 2to 9.5for 1h.Substantial improvement in laccase stability against different pH conditions was observed for the immobilized laccase (Fig.12),especially for the PES-4%TiO 2-GLU-Post membrane,which exhib-ited the best tolerance over a wide range of pH value,while the pure in-house PES membrane displayed the worst.

3.2.

4.Kinetics of the immobilized laccase on TiO 2functionalized membrane

Table 5shows the Michaelis –Menten kinetic constants for different bio-catalytic membranes.K m value for enzyme immobi-lized on the in-house PES membrane was only slightly higher than the free laccase,indicating that physical adsorption could better preserve the af ?nity between enzyme and substrates than cova-lent bonding.In comparison,for both covalently bonded mem-branes,the K m values were around 2.7–2.8times higher than the free laccase.In terms of k cat /K m ,the bio-catalytic membrane with sequential immobilization procedure displayed the best ef ?ciency in converting substrates (ABTS)into the product.These results suggested the sequential immobilization as the preferred immo-bilization process in terms of the reaction kinetics.

The K m values before and after immobilization in both mem-brane system and batch and packed bed reactors are summarized in Table 6.In terms of the packed bed and batch reactors,the K m value of the immobilized laccase was reported to be around 5.5–22.9times higher than that of the free laccase [18,52,53].Such results indicated that enzyme immobilization on the TiO 2functionalized

NaOH MeOH Acetone

20406080100R e s i d u a l a c t i v i t y (%)

PES-4% TiO 2-Seq PES-4% TiO 2-GLU-Post In house PES

Fig.11.Residual activity of in house PES,PES-4%TiO 2-Seq and PES-4%TiO 2-GLU-Post membranes after incubation with different chemical denaturants.

20406080100

R e m a i n i n g a c t i v i t y (%)

pH

Fig.12.Residual activity of free laccase,in house PES,PES-4%TiO 2-Seq and PES-4%TiO 2-GLU-Post membranes (incubation in different pH value buffer solutions for 1h).

Table 5

Kinetic parameters of the enzyme immobilized on TiO 2functionalized membranes.

Free laccase

In house PES PES-4%TiO 2-Seq PES-4%TiO 2-GLU-Post k cat (m mol min à1g à1)496.3721.17.070.522.973.811.170.9K m (m M)

22.372.027.373.260.073.262.374.5k cat K m à1(L min à1g à1)

22.371.8

0.2670.02

0.3870.03

0.1870.02

Table 6

Summary of the changes of K m value before and after immobilization in membrane system and packed bed and batch reactors.Carrier

Reactor

K m (free enzyme)K m (immobilized enzyme)K m (immobilized)/K m (free)Reference PES-4%TiO 2-Seq

Membrane 22.3

60.0

2.7Current work Polypropylene membrane Membrane 0.2070.040.3670.06 1.8[34]p(HEMA-g-GMA)?lm

Membrane 10.0

23

2.3[49]Cellulose-based Granocel-4000Batch reactor

39.471.4214.9710.2 5.5[52]CR633Packed bed reactor 31.072.0711.0715.122.9[18]Silica-gel

Batch reactor

39.471.4

417.2718.5

10.6

[53]

J.Hou et al./Journal of Membrane Science 452(2014)229–240

238

membrane is potentially a better solution to fully utilize the catalytic function of the laccase than the conventional packed bed and batch reactors.The change of K m was also compared between different membrane systems from literature.Immobilization of laccase on polypropylene membrane by covalent bonding was explored by Georgieva et al.[34],and the K m value increase1.8time by using phenol as the substrate for the kinetic study.Bayramoglue et al.[50] reported the K m values increased2.3times after laccase immobilized on p(HEMA-g-GMA)?lm.These results were in good agreement with the results from our work.

4.Conclusion

In this work,laccase was successfully immobilized on both TiO2 nanoparticles and TiO2nanoparticle functionalized PES mem-branes by using different immobilization approaches.The results revealed that both the immobilization procedures and the proper-ties of the immobilization supports have signi?cant impacts on the biocatalyst performance in terms of the laccase activity,loading, activity recovery,stability,and kinetics parameters.

Among three different immobilization procedures,the sequen-tial procedure was considered as the optimum approach for laccase immobilization as it exhibited the highest activity and activity recovery on both particles and membranes.By contrast, GLU post-treatment displayed lower activity and activity recovery even though it gave the best laccase stability due to the formation of the laccase–laccase and laccase-support network structure.Due to the weak bonding formed through van der Waals'force, physical adsorption delivered the lowest stability.

This study also demonstrated that the properties of immobiliza-tion support had strong in?uence on the bio-catalytic performance of immobilized laccase.In terms of the particles,APTES modi?ed TiO2showed better activity compared with non-APTES modi?ed TiO2.Immobilization of laccase on the TiO2functionalized poly-meric membrane combined the bene?ts of both nanoparticles and membrane?ltration system.The laccase loading and activity was strongly in?uenced by TiO2loading,TiO2size and distribution within the membrane matrix.It was proven that4wt%TiO2loading offered the optimized bio-catalytic performance in this work.The results also indicated such a bio-catalytic membrane could with-stand wide pH range and severe?ltration operation.In the current study,TiO2nanoparticles were blended in the membrane matrix, further optimization on TiO2loading and particle distribution on membrane surface is essential to provide the membrane with optimized architecture which enhances the performance of enzyme immobilization.Such a goal can be achieved through the sol–gel deposition techniques,such as dip coating and vapor sol–gel coat-ing which will be investigated in our future study.

The results also indicated that bio-catalytic membrane pre-pared by the sequential procedure could better maintain enzyme's af?nity to the substrate(ABTS)compared with the packed bed and batch reactors.Further kinetic studies using real micropollutants such as bisphenol-A substrate are necessary to examine the feasibility of such a bio-catalytic membrane system.In addition, when applying this bio-catalytic membrane for micro-pollutant(e.

g.BPA)degradation,organic acid and inorganic ions normally co-exist in the wastewater stream.The impact of these components on the enzymatic performance for BPA degradation is also needed for further investigation.

Acknowledgments

The authors acknowledge the funding from the Australian Research Council(DP1095930).One of the authors(Jingwei Hou)is grateful to the?nancial support of China Scholarship Council (CSC)and the University of New South Wales(UNSW). References

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in on at的时间用法和地点用法 完全版

in，on，at的时间用法和地点用法 一、in, on, at的时间用法 ①固定短语： in the morning/afternoon/evening在早晨/下午/傍晚, at noon/night在中午/夜晚, （不强调范围，强调的话用during the night） early in the morning=in the early morning在大清早， late at night在深夜 on the weekend在周末（英式用at the weekend在周末，at weekends每逢周末） on weekdays/weekends在工作日/周末， on school days/nights在上学日/上学的当天晚上， ②不加介词 this, that, last, next, every, one, yesterday, today, tomorrow, tonight，all，most等之前一般不加介词。如， this morning 今天早晨 （on）that day在那天（that day更常用些） last week上周 next year明年 the next month第二个月（以过去为起点的第二个月，next month以现在为起点的下个月） every day每天 one morning一天早晨 yesterday afternoon昨天下午 tomorrow morning明天早晨 all day/morning/night整天/整个早晨/整晚（等于the whole day/morning/night） most of the time （在）大多数时间 ③一般规则 除了前两点特殊用法之外，其他≤一天，用on，＞一天用in，在具体时刻或在某时用at（不强调时间范围） 关于on 生日、on my ninth birthday在我九岁生日那天 节日、on Teachers’Day在教师节 （注意：节日里有表人的词汇先复数再加s’所有格，如on Children’s Day, on Women’s Day, on Teachers Day有四个节日强调单数之意思，on Mother’s Day, on Father’s Day, on April Fool’s Day, on Valenti Day） 星期、on Sunday在周日，on Sunday morning在周日早晨 on the last Friday of each month 在每个月的最后一个星期五 日期、on June 2nd在六月二日 on the second (of June 2nd) 在六月的第二天即在六月二日 on the morning of June 2nd在六月二日的早晨，on a rainy morning在一个多雨的早晨 on a certain day 在某天 on the second day在第二天（以过去某天为参照） 注意：on Sunday在周日，on Sundays每逢周日（用复数表每逢之意），every Sunday每个周日，基本一个意思。 on a school day 在某个上学日，on school days每逢上学日。on the weekend在周末，on weekends每逢 周末。 关于in in June在六月 in June, 2010在2010年六月

常用标点符号用法简表.doc

常用标点符号用法简表 标点符号栏目对每一种汉语标点符号都有详细分析，下表中未完全添加链接，请需要的同学或朋友到该栏目查询。名称符号用法说明举例句号。表示一句话完了之后的停顿。中国共产党是全中国人民的领导核心。逗号，表示一句话中间的停顿。全世界各国人民的正义斗争，都是互相支持的。顿号、表示句中并列的词或词组之间的停顿。能源是发展农业、工业、国防、科学技术和提高人民生活的重要物质基础。分号；表示一句话中并列分句之间的停顿。不批判唯心论，就不能发展唯物论；不批判形而上学，就不能发展唯物辩证法。冒号：用以提示下文。马克思主义哲学告诉我们：正确的认识来源于社会实践。问号？用在问句之后。是谁创造了人类？是我们劳动群众。感情号①！1.表示强烈的感情。2.表示感叹句末尾的停顿。战无不胜的马克思主义、列宁主义、毛泽东思想万岁！引号 ②“ ” ‘ ’ ╗╚ ┐└1.表示引用的部分。毛泽东同志在《论十大关系》一文中说：“我们要调动一切直接的和间接的力量，为把我国建设成为一个强大的社会主义国家而奋斗。”2.表示特定的称谓或需要着重指出的部分。他们当中许多人是身体好、学习好、工作好的“三好”学生。 3.表示讽刺或否定的意思。这伙政治骗子恬不知耻地自封为“理论家”。括号③（）表示文中注释的部分。这篇小说环境描写十分出色，它的描写（无论是野外，或是室内）处处与故事的发展扣得很紧。省略号④……表示文中省略的部分。这个县办工厂现在可以生产车床、电机、变压器、水泵、电线……上百种产品。破折号⑤——1.表示底下是解释、说明的部

分，有括号的作用。知识的问题是一个科学问题，来不得半点的虚伪和骄 傲，决定地需要的倒是其反面——诚实和谦逊的态度。2.表示意思的递进。 团结——批评和自我批评——团结3.表示意思的转折。很白很亮的一堆洋 钱！而且是他的——现在不见了！连接号⑥—1.表示时间、地点、数目等 的起止。抗日战争时期（1937-1945年）“北京—上海”直达快车2.表 示相关的人或事物的联系。亚洲—太平洋地区书名号⑦《》〈〉表示 书籍、文件、报刊、文章等的名称。《矛盾论》《中华人民共和国宪法》《人 民日报》《红旗》杂志《学习〈为人民服务〉》间隔号·1.表示月份和日期 之间的分界。一二·九运动2.表示某些民族人名中的音界。诺尔曼·白求 恩着重号.表示文中需要强调的部分。学习马克思列宁主义，要按照毛泽 东同志倡导的方法，理论联系实际。······

In on at 时间用法及练习

In\ on\ at (time) at 用在具体某一时刻eg at 11:00 at 4:30 在节假日的全部日子里at Christmas 习惯用法at noon at weekends\ at the weekend at night at breakfast\lunch\supper on 具体到某一天；某一天的早晨，中午或晚上on May the first on Sunday morning 对具体某一天的早晨，中午，晚上进行详细的描述on a sunny morning on a windy night 节日的当天；星期on Women?s Day on Monday In 用在年；月；季节in spring in 2012 in August 后面+一段时间表示将来时in two days 习惯用法in the morning\in the afternoon\in the evening “\”以this, that, last, next, some, every, one, any,all开始的时间副词之前的at\on\in 省略在today, tomorrow, yesterday, the day after tomorrow, tomorrow morning，yesterday afternoon，the day before yesterday 之前的介词必须省略 Practice ___ summer ____ 2012 ____ supper ___ 4:00 ___ June the first ___yesterday morning ____ New Year?s Day ___ Women?s Day ___ the morning ____ the morning of July the first ____ 2014 ___ tomorrow morning ____ midnight 1.—What are you doing ____ Sunday? And what is your wife doing ___ the weekend? 2. He?ll see you ____ Monday. And he…ll see your brother ____next Monday. 3. They often go out ___ the evenings. But they don?t go out ____ Sunday evenings. 4. Do you work ____ Fridays? Does she work _____ every Friday? 5. They usually have a long holiday ___ summer. But their son can only have a short holiday ___ Christmas. 6. Paul got married ___ 2010, He got married ___ 9 o?clock ___ 19 May 2010. His brother got married ___ May, 2011. His sister is getting married ___ this year. 1.—When will Mr Black come to Beijing? ---_______ September 5 A. on B. to C. at D. in 2. The twins were born ____ a Friday evening. A. on B. of C. at D. in 3. It?s the best time to plant ____ spring. A. on B. in C. at D.\ 4. ____ the age of twelve, Edison began selling newspaper on train. A. On B. At C. In D.By 5. She has been an English teacher ____ 2000. A. for B. since C. in D.on 6.I have studied English _____ 2003. A. since B. for C. from D.in

常用标点符号用法含义

一、基本定义 句子，前后都有停顿，并带有一定的句调，表示相对完整的意义。句子前后或中间的停顿，在口头语言中，表现出来就是时间间隔，在书面语言中，就用标点符号来表示。一般来说，汉语中的句子分以下几种： 陈述句： 用来说明事实的句子。 祈使句： 用来要求听话人做某件事情的句子。 疑问句： 用来提出问题的句子。 感叹句： 用来抒发某种强烈感情的句子。 复句、分句： 意思上有密切联系的小句子组织在一起构成一个大句子。这样的大句子叫复句，复句中的每个小句子叫分句。 构成句子的语言单位是词语，即词和短语（词组）。词即最小的能独立运用的语言单位。短语，即由两个或两个以上的词按一定的语法规则组成的表达一定意义的语言单位，也叫词组。 标点符号是书面语言的有机组成部分，是书面语言不可缺少的辅助工具。它帮助人们确切地表达思想感情和理解书面语言。 二、用法简表 名称

句号① 问号符号用法说明。？1．用于陈述句的末尾。 2．用于语气舒缓的祈使句末尾。 1．用于疑问句的末尾。 2．用于反问句的末尾。 1．用于感叹句的末尾。 叹号！ 2．用于语气强烈的祈使句末尾。 3．用于语气强烈的反问句末尾。举例 xx是xx的首都。 请您稍等一下。 他叫什么名字？ 难道你不了解我吗？为祖国的繁荣昌盛而奋斗！停止射击！ 我哪里比得上他呀！ 1．句子内部主语与谓语之间如需停顿，用逗号。我们看得见的星星，绝大多数是恒星。 2．句子内部动词与宾语之间如需停顿，用逗号。应该看到，科学需要一个人贡献出毕生的精力。 3．句子内部状语后边如需停顿，用逗号。对于这个城市，他并不陌生。 4．复句内各分句之间的停顿，除了有时要用分号据说苏州园林有一百多处，我到过的不外，都要用逗号。过十多处。 顿号、用于句子内部并列词语之间的停顿。

2时间介词in,on,at的用法

介词in on at 表示时间的用法及区别 Step1 Teaching Aims 教学生掌握时间介词in,on和at的区别及用法。 Step2 Teaching Key and Difficult Points 教学生掌握时间介词in,on和at的区别及用法。 Step3 Teaching Procedures 1.用in的场合后所接的都是较长时间 (1)表示“在某世纪/某年代/特定世纪某年代/年/季节/月”这个含义时，须用介词in Eg: This machine was invented in the eighteenth century. 这台机器是在18世纪发明的。 、 She came to this city in 1980. 他于1980年来到这个城市。 It often rains here in summer. 夏天这里常常下雨。 (2)表示“从现在起一段时间以后”时，须用介词in。(in+段时间表将来) Eg: They will go to see you in a week. 他们将在一周后去看望你。

I will be back in a month. 我将在一个月后回来。 (3)泛指一般意义的上、下午、晚上用in, in the morning / evening / afternoon Eg: They sometimes play games in the afternoon. 他们有时在下午做游戏。 Don't watch TV too much in the evening. 晚上看电视不要太多。（4）A. 当morning, evening, afternoon被of短语修饰，习惯上应用on, 而不用in. Eg: on the afternoon of August 1st & B. 但若前面的修饰词是early, late时，虽有of短语修饰，习惯上应用in, 而不用on. Eg: in the early morning of September 10th 在9月10的清晨； Early in the morning of National Day, I got up to catch the first bus to the zoo. 国庆节一清早，我便起床去赶到动物园的第一班公共汽车。 2.用on的场合后所接的时间多与日期有关 （1）表示“在具体的某一天”或（在具体的某一天的）早上、中午、晚上”，或“在某一天或某一天的上午，下午，晚上”等，须用介

介词in-on-at在表示时间时的用法

介词in, on, at在表示时间时的用法区别 ①in时间范围大（一天以上）如：in Tanuary, in winter, in 1999;泛指在上午，下午，晚上，如：in the morning(afternoon, evening). 习惯用法：in the daytime 在白天。 ②on指在某一天或某一天的上午，下午，晚上，如：on Monday, on Sunday afternoon, on July 1, 1999 ③at时间最短，一般表示点时间，如at six o’clock, at three thirty.习惯用法：at night, at noon, at this time of year. in, on和at在表达时间方面的区别 in 表示在某年、某季节、某月、某周、某天和某段时间 in a year在一年中 in spring 在春季 in September 在九月 in a week 在一周中 in the morning/afternoon/evening 在上午/下午/傍晚 但在中午，在夜晚则用at noon/night on 表示某一天或某一天的某段时间 on Monday 在周一 on Monday afternoon 在周一下午 on March 7th 在3月7日 on March 7th, 1998. 在1998年3月7日 on the morning of March 7th, 1998. 在1998年3月7日上午

at 表示某个具体时刻。 at eight o’clock 在8点钟 at this time of the year 在一年中的这个时候 at the moment 在那一时刻 at that time 在那时 注意：在英语中，如果时间名词前用this, last, next 等修饰时，像这样的表示，“在某时”的时间短语前，并不需要任何介词。 例如：last month, last week, this year, this week, next year, the next day, the next year 等。 1．What’s the weather like in spring/summer/autumn/winter in your country? 你们国家春天/夏天/秋天/冬天的天气怎么样？ in 在年、月、周较长时间内 in a week 在里面 in the room 用某种语言 in English 穿着 in red on 某日、某日的上下午on Sunday afternoon 在……上面 on the desk 靠吃……为生live on rice 关于 a book on Physics 〔误〕We got to the top of the mountain in daybreak. 〔正〕We got to the top of the mountain at day break. 〔析〕at用于具体时刻之前，如：sunrise, midday, noon, sunset, midnight, night。〔误〕Don't sleep at daytime 〔正〕Don't sleep in daytime. 〔析〕in 要用于较长的一段时间之内，如：in the morning / afternoon, 或in the week / month / year. 或in spring / supper /autumn / winter等等。 〔误〕We visited the old man in Sunday afternoon. 〔正〕We visited the old man on Sunday afternoon. 〔析〕in the morning, in the afternoon 如果在这两个短语中加入任何修饰词其前面的介

inonat的时间用法和地点用法版

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日期、onJune2nd在六月二日 onthesecond(ofJune2nd)在六月的第二天即在六月二日onthemorningofJune2nd在六月二日的早晨，onarainymorning在一个多雨的早晨 onacertainday在某天 onthesecondday在第二天（以过去某天为参照） 关于 In 1 2) InJune在六月 inJune,2010在2010年六月 in2010在2010年 inamonth/year在一个月/年里（在将来时里翻译成一个月/年之后） inspring在春天

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inonat的时间用法和地点用法版

i n o n a t的时间用法和 地点用法版 集团档案编码：[YTTR-YTPT28-YTNTL98-UYTYNN08]

i n，o n，a t的时间用法和地点用法 一、in,on,at的时间用法 1、固定短语： inthemorning/afternoon/evening在早晨/下午/傍晚, atnoon/night在中午/夜晚,（不强调范围，强调的话用duringthenight）earlyinthemorning=intheearlymorning在大清早， lateatnight在深夜 ontheweekend在周末（英式用attheweekend在周末，atweekends每逢周末）onweekdays/weekends在工作日/周末， onschooldays/nights在上学日/上学的当天晚上， 2、不加介词 this,that,last,next,every,one,yesterday,today,tomorrow,tonight，all，most等之前一般不加介词。如， thismorning今天早晨 （on）thatday在那天（thatday更常用些） lastweek上周 nextyear明年 thenextmonth第二个月（以过去为起点的第二个月，nextmonth以现在为起点的下个月） everyday每天 onemorning一天早晨 yesterdayafternoon昨天下午 tomorrowmorning明天早晨

allday/morning/night整天/整个早晨/整晚（等于 thewholeday/morning/night） mostofthetime（在）大多数时间 3、一般规则 除了前两点特殊用法之外，其他≤一天，用on，＞一天用in，在具体时刻或在某时用at（不强调时间范围） 关于on On指时间表示： 1）具体的时日和一个特定的时间，如某日，某节日，星期几等。Hewillcometomeetusonourarrival. OnMay4th(OnSunday,OnNewYear’sday,OnChristmasDay),therewillbeacelebra tion. 2)在某个特定的早晨，下午或晚上。 Hearrivedat10o’clocko nthenightofthe5th. Hediedontheeveofvictory. 3)准时，按时。 Iftherainshouldbeontime,Ishouldreachhomebeforedark. 生日、onmyninthbirthday在我九岁生日那天 节日、onTeachers’Day在教师节 （注意：节日里有表人的词汇先复数再加s’所有格，如 onChildren’sDay,onWomen’sDay,onTeachers’Day有四个节日强调单数之意思， onMother’sDay,onFather’sDay,onAprilFool’sDay,onValentine’sDay）星期、onSunday在周日，onSundaymorning在周日早晨

精华版+in,+at,+on表时间的用法

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Don't watch TV too much in the evening. 晚上看电视不要太多。 (4)A. 当morning, evening, afternoon被of短语修饰,习惯上应用on, 而不用in. Eg: on the afternoon of August 1st (5)B. 但若前面的修饰词是early, late时,虽有of短语修饰,习惯上应用in, 而不用on. Eg: in the early morning of September 10th 在9月10的清晨; in the late afternoon of September 12th 在9月12日的傍晚。 Early in the morning of National Day, I got up to catch the first bus to the zoo. 国庆节一清早,我便起床去赶到动物园的第一班公共汽车。 用on的场合后所接的时间多与日期有关 (1)表示“在具体的某一天”或(在具体的某一天的)早上、中午、晚上”,或“在某一天或 某一天的上午,下午,晚上”等,须用介词on。 Eg: Jack was born on May 10th, 1982. 杰克生于1982年5月10日。 They left on a rainy morning. 他们是在一个雨天的早上离开的。 He went back to America on a summer afternoon. 他于一个夏天的下午返回了美国。

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引号“”‘’括号[] 破折号——省略号……书名号 着重号· 间隔号· 连接号— 专名号____ 备注占两格左上角

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位置，它们的前一半可以放在一行的开头，但不出现在一行的末尾，后一半不出现在一行的开头。 破折号和省略号都占两个字的位置，可以放在一行的开头，也可以放在一行的末尾，但不可以把一个符号分成两段。这两种符号的位置都写在行次中间。） 引用之语未独立，标点符号引号外；引用之语能独立，标点符号引号里。 注意事项： 冒号 表示提示性话语之后的停顿，用来提引下文。 ①同志们，朋友们：现在开会了。 ②他十分惊讶地说：“啊，原来是你！” ③北京紫禁城有四座城门：午门、神武门、东华门和西华门。 注意：“某某说”在引语前，用冒号；在引语中或引语后，则不用冒号。如： ⑴老师说：“李白是唐代的大诗人，中学课本有不少李白的诗。” ⑵“李白是唐代的大诗人，”老师说，“中学课本里有不少李白的诗。” ⑶“李白是唐代的大诗人，中学课本里有不少李白的诗。”老师说。

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时间前面in,on at的区别

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时间介词_at_in_on_用法及练习

时间介词(at, in ,on) 的用法与练习 【时间介词记法口诀】: at用在时刻前，亦与正午、午夜连， 黎明、终止和开端，at与之紧接着相伴。 周月季年长时间，in须放在其前面， 泛指一晌和傍晚，也要放在in后边。 on指特定某一天，日期、星期和节日前 某天上下和夜晚，依然要在on后站。 今明昨天前后天，上下这那每之前， at、in、on都不用，此乃习惯记心间。 1. at+night/noon/dawn/daybreak/点钟（所指的时间小于天）： at night( ) at noon( ) at down( ) at daybreak( ) at7:30( ) at 7o’clock ( ) 2. in+年/月/季节/泛指某一天的上morning，下afternoon，晚evening（所指时间大于天）： in 2004（）in March（）in spring（） in the morning（）in the evening （）in the afternoon（） 扩展：在一段时间之后。一般情况下，用于将来时，意为“在……以后”。如：He will come in two hours. 3. on+星期/日期/节日/特指某一天的上，下，晚（所指时间是天）： on Sunday（）on May 4th( )on Sunday morning( ) on Christmas Day( )/on Teachers’ Day( ) on the morning of Sunday( ) on a cold winter morning( ) 扩展：准时，按时。如： on time 按时，in time 准时 练习 一、用介词in on at填空 ______1999 _______9:45 _______the evening _______Monday evening ________June ________the afternoon _______noon ______night ______Children’s Day