Journal of Molecular Catalysis B:Enzymatic45(2007)
84–90
Covalent binding of␣-chymotrypsin on the magnetic
nanogels covered by amino groups
Jun Hong a,b,Dongmei Xu a,b,Peijun Gong a,b,Hanwen Sun a,b,Li Dong a,b,Side Yao a,∗
a Shanghai Institute of Applied Physics,Chinese Academy of Sciences,Shanghai201800,PR China
b Graduate school of Chinese Academy of Sciences,Beijing100039,PR China
Received29June2006;received in revised form24November2006;accepted30November2006
Abstract
A new aminated carrier—magnetic nanogels covered by amino groups,was obtained by Hoffman degradation of polyacrylamide-coated Fe3O4 nanoparticles prepared by photochemical polymerization.␣-Chymotrypsin(CT)was covalently bound to the magnetic nanogels by use of1-ethyl-3-(3-dimethylaminepropyl)carbodiimide and N-hydroxysuccinimide at room temperature.Immobilization time,pH value of the reaction mixture and proportion of CT to the magnetic nanogels were investigated to obtain the optimum condition for CT immobilization.The maximal specific activity of the bound CT w
as determined to be0.93U/(mg min),59.3%of free counterpart.The maximal binding capacity was measured to be 102mg enzyme/g nanogel.Furthermore,the bound CT exhibited good thermal stability,storage stability and reusability.
©2007Elsevier B.V.All rights reserved.
Keywords:Magnetic nanogels;Immobilized enzyme;Photochemical polymerization;␣-Chymotrypsin
1.Introduction
Enzymes,which are usually used as biocatalysts in biochem-ical processes,are preferred to chemical catalyst because they are more selective and of higher efficiency.Immobilization is usually considered to be an important technique to enhance sta-bility of enzymes.Actually,the enzyme stability was greatly dependent on the immobilization strategy[1,2,9].
Recently,a large number of nano-scaled carriers have been applied in the enzyme immobilization[4–10],and enzyme cat-alytic biotechnology is explored for its potential application. Enzyme stability is maximized with nano-scaled supports.How-ever,recovery of the nano-scaled immobilized enzymes from the reaction mixture remains difficult.Considering the facile and fast separation of the magnetic p
articles,magnetic particles are intensively employed as carriers for enzyme immobilization [9,11–15].Using nano-scaled magnetic nanoparticles as the sup-ports of immobilized enzymes are endowed with the following advantages:(1)higher specific area to favor the binding capacity, (2)lower transfer resistance to solve diffusion problem,and(3)∗Corresponding author.Tel.:+862159554681;fax:+862159554681.
E-mail address:yaoside@sinap.ac(S.Yao).readily recovery from the reaction mixture and lower operational cost.However,enzyme immobilization on the non-porous mag-netic particles still bears some shortcomings,for instance,the stability for immobilized enzymes exposition to proteases,inac-tivation by exposition to organic interface[1–3]and denaturation by gas bubble generated in a stirred system[16].
In general,enzymes are covalently bound to supports by use of the reactive groups on the supports[9,17–20].Covalent attachment normally leads to improved enzyme stability,often at the cost of partial deactivation due to the conformational restric-tions imposed by the covalent bonding of enzyme residues to the support.Nowadays,functional magnetic microbeads can be produced in a number of ways but usually involves the coating of magnetically susceptible particles with synthetic polymers having reactive sites for the affinity ligands attachment[21–23].
Photochemical polymerization is a facile“green”method to prepare core-shell composites with different functional groups. The possible mechanism has been proposed in our previous work [28].Properties including particle size and polymeric extent of the magnetic nanogels could be conveniently manipulated by variation of monomer concentration,irradiation time and ratio of magnetite nanoparticles to monomer,etc.The magnetic nanogels could be fast gathered from the reaction system.Most important,the reaction medium is free of initiator and stabilizer.
1381-1177/$–see front matter©2007Elsevier B.V.All rights reserved.
doi:10.lcatb.2006.11.009
MOLCAB-1464;No.of Pages7
J.Hong et al./Journal of Molecular Catalysis B:Enzymatic45(2007)84–9085
Hitherto,the magnetic nanogels with amino or hydroxyl groups
have been synthesized successfully[24,25].
In this study,␣-chymotrypsin(CT)as model enzyme was
covalently bound to the aminated magnetic nanogels by use
of1-ethyl-3-(3-dimethylaminepropyl)carbodiimide(EDC)as
coupling agent.The factors that affected the immobilization
were investigated to obtain the optimum condition for CT immo-
bilization.Furthermore,the reusability,thermal stability and
storage stability of the bound CT were studied.
2.Experimental
2.1.Materials
Acrylamide(AM),N,N -methylene-bis-(acrylamide)
(MBA),1-ethyl-3-(3-dimethylaminepropyl)carbodiimide hy-
drochloride(EDC·HCl),N-hydroxysuccinimide(NHS)and ␣-chymotrypsin(CT)were all of analytic grade and purchased from Shanghai Chemical Reagents Corp.BCA protein assay kit
was available from Beyotime Corp.Fe3O4nanoparticles were
synthesized by partial reduction method according to Refs.
[26,27].They were about10nm in diameter,with a polydisper-
sity index of0.184.
2.2.Synthesis of polyacrylamide-coated Fe3O4 nanoparticles
Polyacrylamide(PAM)-coated Fe3O4nanoparticles were
prepared by photochemical polymerization.0.5g of AM
monomer was dissolved in120ml of water,and mixed with4ml
of1%MBA solution.The mixture wasfiltered with0.45␮mfil-
ter before charging into the quartzflak.The reaction system
was bubbling nitrogen gas to exclude the air inside theflask for
10min,and then2.5ml of magnetite ferrofluid(8mg/ml)was
added.The reaction system was irradiated under500W xenon
lamp for1.5h.After completion of the synthesis,the resultant
nanoparticles were magnetically concentrated and washed sev-
eral times with water.Finally,the sample was vacuum dried and
deposited at desiccator.
2.3.Preparation of magnetic nanogels with amino groups
Twenty milligrams of PAM-coated Fe3O4nanoparticles were
dispersed in100ml of water and sonicated for5min.The
mixture was cooled to about−10◦C and then treated by Hoff-
man degradation.The typical procedures were as follows:a
mixture,which consisted of11.7ml of sodium hydroxide solu-
tion(2.4wt.%)and8.6ml of sodium hypochlorite solution
(5.2wt.%),was cooled at about−10◦C and slowly added into
the suspension of the PAM-coated Fe3O4nanoparticles under
vigorous stirring and the reaction system was kept at ice-salt
bath of−10◦C.After1.5h,58.5ml aqueous solution of sodium
hydroxide(2.4wt.%),cooled to about−10◦C,was slowly
added.The reaction system was kept at ice-salt bath of−10◦C
for0.5h and then at ice-water bath of0◦C for6h.After comple-
tion of Hoffman degradation,the aminated magnetic nanogels
were immediately isolated by a magnet and washed several times with distilled water.The aminated magnetic nanogels were dried under vacuum at room temperature.
2.4.Enzyme immobilization
CT was immobilized onto the magnetic nanogels using EDC as coupling agent.The reaction was carried out under different conditions to determine the optimum condition for ,changes of immobilization time,pH value of the reaction mixture and proportion of CT to the amine-functionalized magnetic nanogels.
For a given pH value of7.4,EDC·HCl(5mg)and NHS(6mg) were dissolved in3ml of phosphate buffer solution(50mM, pH7.4).And then,the amine-functionalized powder(20mg) was added into the above mixture.This system was treated by ultrasonic for10min at0◦C.Subsequently,5mg of CT was added,and then shaken for24h at room temperature.The bound enzyme was collected by an external magneticfield and washed with distilled water for several times.And then,the immobilized enzyme was incubated in PBS(50mM,pH7.4)for30min.This procedure was repeated for several times until no free enzyme was detected in PBS by BCA protein assay[9].All the washing solution was pooled and the total protein concentration was mea-sured using BCA protein assay.Finally,the bound enzyme was dispersed in0.001M of hydrochloric acid solution.The binding capacity was calculated as:
M(mg enzyme/g nanogel)=
m−C1V1
W
where M represented the binding capacity,C1and V1were the concentration and volume of washing solution after immobiliza-tion,respectively,m the weight of enzyme introduced into the immobilization system,and W was the weight of the aminated magnetic nanogels.
2.5.Characterization
The binding of CT on the aminated magnetic nanogels was examined by a Nicolet FT-IR spectrophotometer and a PHILIPS CM120transmission electron microscopy(TEM).The binding capacity was determined with BCA protein assay kit.A stan-dard curve was constructed with BSA.Polymeric extent was estimated by a simultaneous DTA-TG(Shimadzu,DTG-60M) and DSC apparatus(Shimadzu,DSC-60)by heating the sam-ples from room temperature to700◦C under N2atmosphere at
a heating rate of10◦C min−1.
2.6.Enzyme activity assay
Unit of enzyme activity(U)was defined as:1mg of protein will hydrolyze1.0␮mol of BTEE per min at pH7.8at25◦C. Enzyme activity of the bound CT was determined with a UV–vis spectrophotometer(Shimadzu,Model1601;Tokyo,Japan). BTEE+H2O CT
−→N-benzoyl-l-tyrosine+ethanol
The assay mixture was composed of1.42ml of Tris–HCl buffer(80mM,pH7.8),1.4ml of1.18mM BTEE and0.08mlreaction to a book or an article
86
J.Hong et al./Journal of Molecular Catalysis B:Enzymatic 45(2007)84–90
of 2M CaCl 2.After addition of 0.1ml of enzyme solution,the reaction was carried out at 25◦C for 3min.The suspension was immediately separated by an external magnetic field of 0.5T and measured the absorbance of the solution at 256nm.The specific activity was calculated as follows:
specific activity (U /mg min)=
A
0.964×Ew ×3×3
where  A was the absorbance change of the solution at 256nm,Ew represented the amount of enzyme contained in 0.1ml of enzyme solution,0.964was the molar extinction coefficient of N -benzoyl-l -tyrosine at 256nm.2.7.Thermal stability measurement
Thermal stabilities of free and bound CT were checked by measuring their residual activities after being incubated for 30min in the temperature range of 35–85◦C.Thermal stabil-ities of free and bound CT were also examined by assaying their residual activities after being incubated at 45◦C for a required period.All data used in this formula were averages of duplicated experiments.2.8.Storage stability
Activities of free and bound CT after storage in the hydrochlo-ric acid solution (pH 3)at 4or 25◦C were determined by measuring the absorbance at 256nm.The measurements were performed at intervals of a week within a period of 36days.2.9.Reusability assay
The reusability of bound CT was examined by conducting the activity measurement of bound CT at 25◦C at time intervals of 30min.After each activity measurement,the bound CT was sep-arated magnetically and washed several times with PBS.Then,1.42ml of Tris–HCl buffer (80mM,pH 7.8),1.4ml
of 1.18mM BTEE and 0.08ml of 2M CaCl 2were added to the bound CT in sequence and the next activity measurement was carried out.
3.Results and discussion
3.1.Preparation of the magnetic nanogels covered by amino groups
In this study,magnetic nanogels containing reactive amino groups were obtained by Hoffman degradation of PAM-coated Fe 3O 4nanoparticles,prepared by photochemical polymeriza-tion with quantum-sized Fe 3O 4nanoparticles as photoinitiator.Since amino-functionalized magnetic nanogels tended to aggre-gate,Hoffman degradation was optimized in order to obtain magnetic nanogels with smaller particle size.In the experiment,the solution of sodium hydroxide and sodium hypochlorite was slowly added into the reaction mixture,aiming at minimizing aggregation caused by fierce reaction.The reaction was carried out at low temperature to lower the rate of reaction and prevent
the magnetic nanogels against aggregation.On the other hand,side reactions such as hydrolysis of amido groups to form alky-lacrylureas and alkylureas and the cleavage of the hydrocarbon main chain would take place during the Hofmann degradation of PAM.These reactions could be effectively prevented if the reaction temperature was low enough [37].As amino groups tended to be oxidized [36],
N 2was hereby bubbled as protective gas throughout the experiment.It was proved that the mag-netic nanogels with amino groups were successfully obtained [27].
The amine-functionalized magnetic nanogels were about 25nm in hydrodynamic diameter.Combined with the result of conductometric titration and the polymeric extent of PAM-coated Fe 3O 4nanoparticles (10.8%),the amination degree of the aminated magnetic nanogels was determined to be 74.8%,namely,74.8%of amido groups converted to amino groups.There were some uneliminated carbonyl groups on the mag-netic nanogels,otherwise further Hoffman elimination would destroy surface structure of the magnetic nanogels.Magnetic content of Fe 3O 4was as high as 80%,which guaranteed that the magnetic nanogels were susceptive to external magnetic field.
3.2.Binding of CT on the magnetic nanogels with amino groups
3.2.1.Immobilization
CT was covalently bound on the amine-functionalized mag-netic nanogels via carbodiimide activation in the PBS (50mM,pH 7.4).In this coupling reaction,active ester was formed between CT and NHS,and then reacted with amino groups exist-ing on the magnetic nanogels.The immobilization protocol was as illustrated in Fig.1.
3.2.2.Confirmation of CT bound to the magnetic nangels with amino groups
The binding of CT onto the magnetic nanogels was con-firmed by TEM observation and FT-IR spectrum measurement.In order to keep morphology of the magnetic nanogels,the sample of TEM was freeze dried at liquid nitrogen (−196◦C)and then vacuum dried.As was evident from Fig.2,CT was layered over the amine-functionalized magnetic nanogels.The bound CT possessed considerable dispersancy.However,aggregation occurred due to interaction between magnetic nanoparticles.Fig.3showed the FT-IR spectral characteris-tics of CT bound to the magnetic nanogels.The peaks of 1637.1cm −1,1530.4cm −1and 1399.4cm −1,which also existed in the IR spectrum of the bound CT,were the characteristic peaks of CT.Strong absorption band around 578.6cm −1was ascribed to the Fe–O bond of naked Fe 3O 4.Additionally,the sample of bound CT for FT-IR spectrum measurement was washed with distilled water,and then incubated in PBS until no free enzyme was detected by BCA protein assay before being dried.This procedure guaranteed that no free enzyme was adsorbed on the support.Consequently,the results above demon-strated clearly that CT was bound to the magnetic nanogels successfully.
J.Hong et al./Journal of Molecular Catalysis B:Enzymatic 45(2007)84–90
87
Fig.1.Schematic presentation of CT immobilization on the magnetic
nanogels.
Fig.2.TEM images of (a)uncoated Fe 3O 4nanoparticles,(b)magnetic nanogels with amino groups and (c)bound CT.
3.2.3.Effect of pH value of buffer solution on the immobilization
It is well known that pH has a crucial importance on the enzymes’properties.For extreme situation (inadequate pH value or long term exposure to medium of inadequate pH),enzymes will permanently loss their activities [32].
Considering hydrolysis of active esters and influence of pH on the activity of enzyme,the coupling reaction was carried out in the pH range of 5.8–8.0.The binding capacity was deter-mined by estimating the enzyme in the washing.As shown in Table 1,when the reaction was performed at pH 8.0,the mag-netic nanogels did not show enzyme activity,and no enzyme was detected by BCA protein assay.Therefore,it was rea-sonable to assume that no enzyme was bound at pH 8.0.At pH 7.4,24.4%of CT was bound to the magnetic nanogels,while 40.8%of CT was immobilized at pH 5.8.The lower pH value of buffer helped to immobilize enzyme onto the mag-netic nanogels.This was in good agreement with the coupling
reaction.
Fig.3.FT-IR spectra of (a)free CT and (b)bound CT.
88J.Hong et al./Journal of Molecular Catalysis B:Enzymatic45(2007)84–90 Table1
Effect of pH on the binding of CT on the magnetic nanogels with amino groups
in3ml of PBS containing5mg of CT
pH of buffer solution Extent of
binding(%)
Binding capacity(mg
protein/g nanogels)
Specific
activity
U/(mg min)
5.840.81020.47
6.538.0950.56
7.424.4610.93
8.000–
Maximal specific activity was observed when the immobi-lization was carried out at pH7.4.Although maximal binding capacity was obtained at pH5.8,only29.9%of the specific activ-ity of the enzyme was retained(Table1).The loss in enzymatic activity might be contributed to the alternations in the propertie
s of the enzyme such as changes in conformation(including the changes caused by pH and immobilization),transfer limitation and so on.The optimum pH for CT binding was observed at pH6.5,which was in agreement with the results that the cou-pling reaction carried out at slightly acidic pH increased the percentage binding of enzyme[34].
3.2.
4.Effect of ratio of CT to the magnetic nanogels on the immobilization
Effect of ratio of CT to the magnetic nanogels with amino groups was investigated.The reaction was performed in3ml of PBS(pH6.5,50mM)for24h at room temperature.As antici-pated,with the higher proportion of CT to the magnetic nanogels, the binding capacity was higher.However,the increase extent of binding capacity decreased when the ratio was above0.3(Fig.4). It was probably related to the limited amino groups on the sur-face of magnetic nanogels exposed to active ester existed in the reaction mixture.
3.2.5.Effect of immobilization time on the immobilization
By assaying the unbound enzyme in the washing solution, effect of immobilization time on the binding capacity was stud-
ied.As indicated in Fig.5,the maximal binding capacity
was
Fig.4.Effect of ratio of CT to the magnetic nanogels on the binding capacity. The reaction was carried out in3ml of PBS(pH6.5,50mM)containing20mg of amine-functionalized magnetic nanogels at room
temperature.Fig.5.The time course of CT immobilization on the magnetic nanogels.The immobilization was carried out in3ml of PBS(pH7.4,50mM)containing 5mg of CT and20mg of amine-functionalized magnetic nanogels at room temperature.
determined to be61mg enzyme/g nanogels;the binding capac-ity increased with increasing the immobilization time;the extent of CT immobilization on the magnetic nanogels almost kept the same when the immobilization time was above12h.This might be related to hydrolysis of active ester in the aqueous solution. With the immobilization time prolonged,the residual active ester in the reaction mixture for enzyme immobilization decreased. Therefore,the binding capacity nearly kept the same when the immobilization time was above12h.
3.3.Properties of the bound CT
3.3.1.Thermal stability
Thermal stability of immobilized enzyme and free enzyme preparations was checked by measuring their residual activities after predetermined thermal inactivation.As seen from Fig.6a, almost no activity was retained for free enzyme when incubation temperature was above75◦C.However,the activity of the bound CT still had a residual activity of88.7%at85◦C.Furthermore, residual activity of the immobilized e
nzyme after an incubation period of4.5h at45◦C was as high as92.5%(Fig.6b),which was higher than that of the free enzyme(85.6%).This resulted suggested that thermostability of CT became higher than that of free CT at high temperature,in good agreement with the results previously reported[29–31].This might be due to the covalently bound enzyme being protected from conformational changes caused by heat.
3.3.2.Storage stability
In order to investigate the industrial practicability of an immo-bilized enzyme,the loss in enzyme activity,known as storage stability,is an important parameter to be taken into account. As presented in Fig.7,free CT stored at25◦C lost its all-initial activity within22days,while free enzyme stored at4◦C retained 83.5%of its activity after a storage period of36days.The immo-bilized enzyme stored at4◦C nearly kept their all-initial activity, yet the immobilized enzyme stored at25◦C only lost about10% of its activity after a36-day storage.The covalent immobiliza-tion definitely held the enzyme in a stable position in comparison
J.Hong et al./Journal of Molecular Catalysis B:Enzymatic45(2007)84–90
89
Fig.6.Thermal stabilities of free and immobilized CT(a)incubated in the temperature range of35–85◦C for30min and(b)incubated at45◦
C.
Fig.7.Storage stabilities of free and bound CT.
to the free counterpart[33].On the other hand,hydrophobic group containing supports minimized possible distortion effects imposed from aqueous medium on the active site of the immo-bilized enzyme[34,35].The experiment revealed that storage stability of the bound CT was improved in comparison to free CT.
3.3.3.Reusability
Reusability of immobilized enzymes was important for their practical application.As shown in Fig.8,the activity of bound CT had no significant loss after being reused six times within2h. This indicated the resultant bound CT had excellent reusability, which was desirable for applications in biotechnology.
4.Conclusions
The magnetic nanogels,covered by hydrophilic coating layer containing reactive amino groups were obtained by Hoffman degradation of PAM-coated Fe3O4nanoparticles prepared by photochemical polymerization.CT was covalently bound to the magnetic nanogels via carbodiimide activation at room
tem-
Fig.8.Reusability of bound CT.
perature.The optimal conditions for enzyme immobilization depended on the immobilization time,the pH of the reaction mixture and the ratio of enzyme to magnetic nanogels.Lower pH value of buffer helped to immobilize enzyme onto the mag-netic nanogels.Meanwhile,more loss in activity of the bound CT was observed.Higher proportion of CT to the magnetic nanogels helped to enhance the binding capacity.With increasing the immobilization time,the binding capacity increased,and the extent of CT immobilization on the magnetic nanogels almost kept the same when the immobilization time was above12h. After being bound,CT had a maximal residual activity of59.3% of the free enzyme,and exhibited good thermal stability,storage stability and reusability.
Acknowledgements
This work wasfinancially supported by Science&Technol-ogy Commission of Shanghai Municipality(No.0452nm068) and National Natural Science Foundation of China(20504010). Many thanks to Professor Li Yuan.

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