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Journal of Magnetism and Magnetic Materials 322 (2010) 2031–2037

Contents lists available at ScienceDirect

Journal of Magnetism and Magnetic Materials

0304-88

doi:10.1

n Corrnn Cor

E-m

(H. Che1 Th

journal homepage: www.elsevier.com/locate/jmmm

Immobilized protease on the magnetic nanoparticles used for the hydrolysisof rapeseed meals

Xin Jin a,1, Ju-Fang Li b,1, Ping-Ying Huang a,1, Xu-Yan Dong a,b, Lu-Lu Guo b, Liang Yang a,Yuan-Cheng Cao a, Fang Wei b, Yuan-Di Zhao a,n, Hong Chen b,nn

a Wuhan National Laboratory for Optoelectronics—Hubei Bioinformatics and Molecular Imaging Key Laboratory, College of Life Science and Technology, Huazhong University of

Science and Technology, Wuhan, HuBei 430074, PR Chinab The Key Lab of Oil Crops Biology, Ministry of Agriculture, Institute of Oil Crops Research, Chinese Academy of Agricultural Sciences, Wuhan, Hubei 430062, PR China

a r t i c l e i n f o

Article history:

Received 2 November 2009

Received in revised form

6 January 2010Available online 25 January 2010

Keywords:

Magnetic nanoparticle

Immobilized protease

Rapeseeds meal

Hydrolysis

53/$ - see front matter & 2010 Elsevier B.V. A

016/j.jmmm.2010.01.029

esponding author. Tel.: +86 27 87792235; fa

responding author. Tel.: +86 27 86812943; f

ail addresses: zydi@mail.hust.edu.cn (Y.-D. Z

n).

ese authors made equal contribution to the

a b s t r a c t

(3-aminopropl) triethoxysilaneand modified magnetic nanoparticles with the average diameter of

25.4 nm were synthesized in water-phase co-precipitation method. And then these nanoparticles were

covalently coupled with alkaline protease as enzyme carrier by using 1,4-phenylene diisothlocyanate as

coupling agent. Experiments showed that the immobilized protease can keep the catalytic bioactivity,

which can reach to 47.8% when casein was served as substrate. Results showed that the catalytic

activity of immobilized protease on these magnetic nanoparticles could retain 98.6372.37% after 60

days. And it is more stable than the free protease during the shelf-life test. The enzyme reaction

conditions such as optimum reaction temperature and pH are the same as free protease. Furthermore,

mix-and-separate experiments showed that the immobilized protease could be recycled through the

magnetic nanoparticles after the biocatalysis process. When the rapeseed meals were used as substrate,

the degree of hydrolysis of immobilized alkaline protease achieved 9.86%, while it was 10.41% for the

free protease. The macromolecular proteins of rapeseed meals were hydrolyzed by immobilized

protease into small molecules such as polypeptides or amino acids. Thus, a novel efficient and economic

way for the recycling of enzymes in the application of continuous production of active peptides was

provided based on these magnetic nanoparticles.

& 2010 Elsevier B.V. All rights reserved.

1. Introduction

Enzymes are natural biocatalyst with very high catalyticefficiency and selectivity which are widely used in many areassuch as food processing and medical science. However, the stabilityof these enzymes is feeble, and there are still challenges in therecycling of the valuable enzymes, which limit their applications inindustrial production. Not until recently, the immobilized enzymetechnologies overcome these difficulties by immobilizing enzymesonto carriers through physical or chemical methods. Immobilizedenzymes maintain natural catalytic activities and can be easilyseparated from reaction systems for recycling use. Consequently,immobilized enzyme technologies provide a feasible way forcontinuous production [1]. A large number of research reportsshowed that immobilized enzymes have better performance thanfree enzymes in many respects [2–4].

ll rights reserved.

x: +86 27 87792202.

ax: +86 27 86812862.

hao), chenhong@oilcrops.cn

paper.

Various methods have been used to immobilize enzymes, such asadsorption-crosslinking [5], encapsulation [6], covalent coupling [7]and so on. Among these methods, covalent coupling attracts wideattention due to the stability of combination between carriers andenzymes. The carriers designed for the enzymes can be polymers [8],inorganic materials [9] or composites [10]. As magnetic nanoparti-cles can be separated from the reaction systems by applyingexternal magnetic field [11] and therefore these nanoparticles havebeen widely used for biological and medical researches [12], forexample, separation of stem cells [13], immobilization of lipase [14].Such magnetic carriers are generally of core-shell structure. And theshells can be modified with various functional groups (such ashydroxyl, amino, sulfhydryl and so on) by copolymerization orchemical surface modification. And the enzymes, antibodies, nucleicacids are then covalent coupled to the magnetic nanoparticlesthrough organic or inorganic surface modified shells.

Meanwhile, the rapeseed protein is a kind of vegetable protein,which has a nutritive value no less than that of the animal protein.Rapeseed peptide, degraded from rapeseed meals or rapeseedproteins, is a type of small-molecule peptide with solubility,emulsifying properties and water holding capacities which arebetter than rapeseed protein. In addition, rapeseed peptide has

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several excellent nutritional physiology properties such as fastabsorption and high absorption rate [15]. Thus, it is an importanteconomic resource for the high value rapeseed peptide fromrapeseed meals. The enzyme method to obtain peptides byhydrolyzing protein attracts considerable attention because ofits mild reaction conditions, safety, controllability, and thecapability of large-scale production of specific peptides [16].Alkaline protease is a kind of proteolytic enzyme produced byfermenting bacillus subtilis 2709, and its catalytic site is serine[17]. Alkaline protease exhibits outstanding capability to degradeprotein, and has many free amino acids which serve as the activesites for covalent conjugation.

In this study, amino silane coated magnetic nanoparticles weresynthesized in water phase, then alkaline proteases wereimmobilized onto the surface of these magnetic nanoparticlescarriers with 1,4-phenylene diisothlocyanate as coupling agent.The resultant immobilized protease was used for biocatalysis fordifferent kinds of proteins in rapeseed meals, and it can beseparated easily by applying external magnetic field. This workprovides a promising way to recycle the enzymes and alsoprovides a feasible way to produce active peptides continuouslyby mix-and-separate.

2. Materials and methods

2.1. Material and reagents

FeCl3 �6H2O, FeSO4 �7H2O, aqueous solution (25%) and N,N-dimethylformamide (DMF) were purchased from SinopharmChemical Reagent Co. Ltd. (Shanghai, China). (3-aminopropl)triethoxysilaneand (APTES, 98%), 1,4-phenylene diisothlocyanate(PDC, 98%) were obtained from Alfa Aesar (Ward Hill, USA).Fluorescamine (99%) was provided by Sigma-Aldrich Fine Chemi-cals (St. Louis, USA). All reagents were of analytical grade.

Casein was purchased from Huashun Chemical Reagent Co.Ltd. (Wuhan, China), which was of chemical grade. The alkalineprotease (50,000 U/g) was biochemical reagent provided byHuashun Chemical Reagent Co. Ltd. (Wuhan, China).

2.2. Immobilizing of alkaline protease onto magnetic nanoparticles

Magnetic nanoparticles were synthesized by co-precipitation[12] in water phase. A mixture solution of ferric and ferrous ions ina 1:2 molar ratio was prepared in flask under argon atmosphere,and the pH was adjusted to 1.7 by HCl solution (0.1 M). And then,ammonia aqueous solution (5%) was dropped into flask withviolently stirring till the pH rose to 9.0 and then kept stirring for0.5 h. The resultant Fe3O4 nanoparticles were separated by externalmagnetic field, and then washed with water for 5 times and twicewith ethanol. Finally, the magnetic Fe3O4 nanoparticles weredispersed in ethanol with the concentration of 3.45 mg/mL (about0.015 M).

25 mL colloidal Fe3O4 ethanol solution prepared above wasdiluted with 75 mL water and 50 mL ethanol, and then purgedwith argon for at least 10 min. In order to disperse completely, thesolution was ultrasonically agitated for 0.5 h and a certain amountof APTES was added. After that, the mixture solution was keptstirring for 40 h before separation. The surface modified magneticnanoparticles (APTES-Fe3O4) were washed with ethanol for 5times, and then dispersed in 25 mL ethanol with the concentra-tion of 3.60 mg/mL.

Then alkaline proteases were conjugated with these magneticnanoparticles by covalent coupling using PDC as coupling agent.In brief, the APTES-Fe3O4 nanoparticles were dispersed in DMF,

the same volume of PDC–DMF solution (2 mg/mL) was added andshaked for 3 h. After that, the nanoparticles were washed by DMF,methanol, acetone, borate buffer solution (0.2 M, pH 10.5) once,respectively. Then a certain amount of alkaline protease solution(dispersed in borate buffer solution, 0.6 U/mL) was added and theresultant was shaked overnight. At last, the immobilized pro-teases (E-APTES-Fe3O4) were washed with borate buffer solution(0.2 M, pH 10.5) for several times and dispersed in borate buffersolution with the concentration of 8.50 mg/mL.

2.3. The enzyme assays of the immobilized protease

Some important reaction conditions of both immobilizedprotease and free protease were optimized. 1 mL immobilizedprotease solution was heated to the reaction temperature in waterbath for 2 min, then 1 mL casein solution (10 mg/mL, dissolutted inborate buffer at certain pH) was added. After that, the mixture wasincubated at certain temperature for 10 min. 2 mL trichloroaceticacid was added immediately in order to stop the reaction, and then,the mixture was centrifuged at 5000 rpm for 10 min. The catalyticactivity of the supernatant was determined by the Folin-phenolmethod, using casein as substrate [10]. 1 mL supernatant wasmixed with 5 mL Na2CO3 solution (0.4 mol/L) and 1 mL Folin-phenol reagent (diluted once). The mixture was kept at 40 1C for20 min, the absorbance of which was measured at 680 nm. Finally,the catalytic activity was calculated according to the equation:X=A�K�4/10�n (X—the catalytic activity of the samples (IU/g orIU/mL), A—the absorbance, K—absorption constant, 4—the volumeof the reaction mixture (mL), 10—the reaction time, n—dilutionmultiple). Different temperature levels from 25 to 75 1C (10 1Cinterval) were set to determine the optimal temperature at fixedpH 10. The optimal pH of catalytic activity was tested in differentpH borate buffer selected from 6 to 12 (1 interval) at fixedtemperature 40 1C. The storage stability was observed by measur-ing the catalytic activity of the same amount of immobilized andfree proteases dispersed in borate buffer solution at the optimumtemperature and pH in a fixed interval time during a period of 60days. The test samples were kept at 4 1C during this period.

The degree of hydrolysis (DH) of rapeseed meals was analyzedwith ninhydrin [18]. 50 mg rapeseed meals were dispersed in3 mL phosphate buffer solution (pH 10.5). In order to make themolecules to unfold and become more accessible for hydrolyticreaction [19], the proteins were denatured first and then thesuspension liquid was kept at 80 1C for 30 min. Then, 2 mLimmobilized protease was added and reacted at 50 1C for 2 h.After that, the mixture was boiled for 15 min to deactivate theprotease activity, centrifuged at 5000 rpm. 2 mL diluent suspen-sion was mixed with 1 mL ninhydrin ethanol solution, incubatedin boiling water for 15 min. After that, the mixture was cooled toroom temperature, then 5 mL ethanol solution (45%) was added.The absorbance of the resultant solution was measured at 570 nmafter standing for 15 min. The degree of hydrolysis of rapeseedmeals was calculated with the equation: DH=(the amino nitrogencontent of hydrolysate/the total amino nitrogen content ofrapeseed meals)�100%

Electrophoresis was performed using 16.5% acrylamide separa-tion gels, 10% acrylamide spacer gel and 4% acrylamide stackinggels under denaturing and reducing condition. Reduction wasachieved by heating the sample in boiling water for 5 min in thepresence of 2-mercaptoethanol (10%, v/v).

2.4. Characterization

The crystal structure of the particles was determined by X’PertPRO X-ray diffraction (PANalytical B.V., Almelo, Netherlands) with

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high-intensity Cu radiation (l=0.154060 nm). TEM-100CXII trans-mission electronic microscopy (JEOL, Tokyo, Japan) was used todescribe the particle size and morphology. The change of chemicalbond between APTES and Fe3O4 was studied by VERTEX 70Fourier transform infrared spectroscopy (Bruker, Karlsruhe,Germany). The elemental analysis before and after APTES coatingwas studied by EAGLE III X-ray fluorescence (EDAX Inc., Mahwah,USA). Vibrating sample magnetometer model 4HF (ADE Corp.,Milpitas, USA) was used to measure the magnetic performance ofthe samples. The fluorescence spectra of fluorescamine weretested with a LS-55 luminescence spectrometer (Perkin-Elmer,Waltham, USA). The UV–visible absorption spectra was obtainedfrom a UV-2550 UV–vis spectrophotometer (Shimadzu, Kyoto,Japan) when detecting the catalytic activity of immobilizedprotease.

The average size of the nanoparticles was estimated by Debye–Scherrer equation

d¼Kl

bcosð2y=2Þð1Þ

In this equation, d is the average particle size (nm), l is the X-raywavelength (l=0.154 nm), K is the constant (K=0.890), b is thefull width at half maximum (FWHM) of the most intense (3 1 1)peak (b=0.00467 rad), and y is the corresponding diffraction angle[20].

3. Results and discussions

3.1. Characterization of the composite nanoparticles

The X-ray diffraction (XRD) pattern was measured first toconfirm the crystal structure of the bare Fe3O4 particles (Fig. 1).The main diffraction peaks of the sample were located at2y=30.41591, 35.81361, 43.39781, 53.82131, 57.32401 and62.93441, corresponding to (2 2 0), (3 1 1), (4 0 0), (4 2 2), (5 1 1)and (4 4 0) crystal face of inverse spinel Fe3O4 with a face-centered cubic phase, which are consistent with the characteristicdiffraction peaks of normative Fe3O4 [21]. The XRD patternindicated the nanoparticles obtained was Fe3O4 withcomparatively high purity. Therefore, the size of the bare Fe3O4

nanoparticles was calculated as 25.4 nm.Transmission electronic microscopy (TEM) photography was

taken after Fe3O4 had been modified with APTES (Fig. 2). Thediameter of the Fe3O4 coated with APTES (Fig. 2B) increased a

Fig. 1. XRD pattern of magnetic nanoparticles.

little, compared with bare Fe3O4 (Fig. 2A). The average diameter ofbare Fe3O4 was about 25.4 nm, but for APTES-Fe3O4 it is about30.0 nm. From the results we can see that the APTES modificationdoes not change the surface morphology of Fe3O4 nanoparticles;however, this surface modification improved solubility andavoided aggregation.

This modification procedure was confirmed by comparison ofFourier transform infrared spectroscopy (FT-IR) spectra of bareFe3O4 and APTES-Fe3O4. In Fig. 3(a), bands in 3422.74 and1628.83 cm�1 were due to the stretching vibration and bendingvibration of –OH, respectively, bands in 575.09, 792.83,888.86 cm�1 referred to stretching vibration of Fe–O–Fe [22]. Asshown in Fig. 3(b), the band in 3435.10 cm�1 might becontributed by the stretching vibration of –OH and –NH2, and itwas hard to determine the existence of –NH2. However, bands in

Fig. 2. TEM photography of bare Fe3O4 (A) and Fe3O4 coated with APTES (B).

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1115.98 and 1017.22 cm�1 referring to stretching vibration ofSi–O might give evidence of the existence of APTES [23]. Inaddition, bands in 586.10, 795.69, 891.58 cm�1 are also due tostretching vibration of Fe–O. All of these results revealed that thenanoparticles were successfully modified with the APTES throughchemical bond.

X-ray fluorescence (XRF) elemental analysis of bare andmodified Fe3O4 showed that, bare Fe3O4 only contained Fe andMn. However, after the modification of APTES, the content of Si inthe samples increased to 4.12%, which suggested that Fe3O4 wassuccessfully coated by APTES.

The presence of amino group (–NH2) on the surface of APTES-Fe3O4 could be verified by fluorescamine which will change intofluorescent. As shown in Fig. 4A (curve a), there was nofluorescence in the control fluorescamine solution (withoutAPTES-Fe3O4), which indicated that there is no amino group inthis blank solution [24]. After the addition of APTES-Fe3O4

nanoparticles, the fluorescence could be detected at 480 nm.

Fig. 3. FT-IR spectra of the bare Fe3O4 (a) and APTES-Fe3O4 (b).

Fig. 4. The fluorescence spectrum of fluorescamine reacted with different concentration

from a to g: 0, 0.12, 0.24, 0.36, 0.60, 0.84 and 1.20 mg/mL, respectively; (B) the linear

Further experiments showed that the fluorescence intensityincreased linearly with the amount of the added APTES-Fe3O4

nanoparticles in a certain range (Fig. 4B, R=0.995). The resultsdemonstrated the existence of amino group on the surface ofAPTES-Fe3O4, and these also proved that Fe3O4 nanoparticles weresuccessfully modified by APTES.

The magnetic property of Fe3O4, APTES-Fe3O4 and E-APTES-Fe3O4 were investigated with a vibrating sample magnetometer(VSM). Fig. 5 presented the magnetic hysteresis curves of thesamples. Both the remanence (Br) and coercivity (Hc) of bareFe3O4, APTES-Fe3O4 and E-APTES-Fe3O4 were negligibly small,similar to superparamagnetism. The saturation magnetizationdeclined slightly from 42.7 to 39.7 emu/g with Fe3O4 modified byAPTES. Therefore, the results showed that the APTES-Fe3O4

maintained the magnetic characteristics of bare Fe3O4. Due tocovalent coupled with alkaline protease, the saturationmagnetization of E-APTES-Fe3O4 was changed to 22.3 emu/g. Itis demonstrated that the magnetic property had been slightly

of APTES-Fe3O4: (A) the fluorescence spectrum, the concentration of APTES-Fe3O4

fitting curve of the peak of the fluorescence spectrum.

Fig. 5. Room-temperature magnetization curve of Fe3O4 (a), APTES-Fe3O4 (b) and

E-APTES-Fe3O4 (c).

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changed after surface modifications such as proteaseimmobilization, but it will not affect the final separation in themagnetic field as showed in later experiments.

Fig. 6. The enzyme activity of immobilized protease with different volume

protease used for immobilization (The concentration of protease and APTES-Fe3O4

were 0.6 U/mL and 36.0 mg/mL, respectively).

3.2. The properties of the immobilized protease

A series of experiments were performed to investigate thecatalytic activities of four different conditions, using casein assubstrate (Table 1). The catalytic activity of immobilized proteaseon bare Fe3O4 with PDC was rather low (4 U/mL). That is becauseof lacking functional groups such as amino, the surface of bareFe3O4 cannot immobilize alkaline protease efficiently. Due to thephysical absorb of a very limited amount of protease, the sameresults happened for APTES-Fe3O4 without the addition of PDC(165 U/mL). In addition, experiments showed that if the APTES-Fe3O4 nanoparticles were only treated by PDC, there was also nocatalytic activity was found. While under the same condition, thecatalytic activity of immobilized protease on APTES-Fe3O4 was2875 U/mL. From these results, it certainly indicated that the highactivity of alkaline protease on APTES-Fe3O4 nanoparticles cannotbe the results of physically absorbed enzymes, but the covalentcoupled enzyme on the surface of APTES-Fe3O4 with PDC ascoupling agent. And the results further proved that theimmobilized protease still keeps the biocatalytic activity afterthe immobilization.

Furthermore, the catalytic activities of different amounts ofalkaline proteases immobilized on a certain amount of magneticcarriers were examined. The catalytic activity of immobilizedprotease gradually increased as more alkaline proteases wereadded in the coupling reactions, but it reached the saturation atlast (Fig. 6). The hypothesis is that the active sites for theconjugation on the surface of APTES-Fe3O4 are limited, and so theamount of proteases can be immobilized is also limited. As aresult, the catalytic activity of the immobilized proteases wouldalso reach to the saturation limitation. Thus, when 700 mLproteases was added, the catalytic activity of the immobilizedprotease had already reached saturation limitation, nearly3000 U/mL, and the recovered activity was up to 47.8%. And soin a word, the immobilized protease obtained has very highcatalytic activity. The catalytic activity of immobilized protease islower than the free protease, and the reason is probably that someactive domains of protease were blocked or reformatted after theconjugation. In addition, the existence of Fe3O4 would also affectthe catalytic activity of immobilized protease due to the change ofspace freedom [25].

To further confirm the characters of the immobilized protease,the optimum reaction temperature of both immobilized and freeprotease was also studied. The catalytic activity of bothimmobilized and free protease is increasing as temperatureincreased until it reached the maximum at 65 1C. However, itdeclined rapidly as temperature continually increased. It wasproved that the optimum reaction temperature of both immobi-lized and free protease is between 60 and 65 1C, and the optimumreaction temperature of immobilized protease is appreciablyhigher than that of the free protease.

Then, the optimum reaction pH of both immobilized and freeproteases was tested. It could be seen that the catalytic activity ofimmobilized and free proteases were increasing as pH increased,

Table 1The catalytic activity of four different conditions.

Samples Fe3O4+PDC+E APTES-Fe3O4+

Catalytic activity 4 U/mL 0

which reached the saturation until the pH value was 10.0. Afterthat, the catalytic activity decreased as the increasing of pH. Theoptimum reaction pH value of both immobilized and free proteasebasically is the same, around 10.0. Between pH 8.0–10.0, they areable to maintain relatively high catalytic activity.

The shelf-life of immobilized protease is another importantaspect of its applications; further experiments were carried out toevaluate its storage stability. The catalytic activity of the sameamount of immobilized and free proteases dispersed in borate buffersolution kept at 4 1C was measured during a period of 60 days(Fig. 7). Compared by averages and standard deviations, the catalyticactivity of immobilized protease maintained 98.6372.37% after 60days, while 93.9373.05% for free protease. This indicated that thestorage stability of immobilized protease was slightly better thanfree protease during the test period. Finally, pair T test of these twowas carried out, and the results proved that the difference of thestabilities between immobilized and free protease is significant(T13=5.615, Po0.01), and which is not affected by measuredeviations. From these results, it can be seen that the immobilizedprotease retained better catalytic activity and kept relatively morestable bioactivity for long shelf-time.

Then, the ability to separate and recycle immobilized proteasewas tested. After immobilized protease had reacted with casein, itwas separated from the reaction solution by magnetic field. Theresults demonstrated that immobilized protease can be separatedsuccessfully and it has good recycling capacity.

Immobilized protease was used to enzymatic hydrolyze rape-seed meals to further investigate its catalytic properties. Theprotein content of rapeseed meals was 42.7% with 2S/1.7Salbumin as its main components. The electrophoretograms oftheir hydrolysate were shown in Fig. 8. Proteins of rapeseed mealscontaining many macromolecular proteins showed a widedistribution (Fig. 8, channel IV) which can be proved byelectrophoretograms of 2S albumin (Fig. 8, channel II). After theaddition of free protease, the bands for macromolecular proteins

PDC APTES-Fe3O4+E APTES-Fe3O4+PDC+E

165 U/mL 2875 U/mL

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of both rapeseed meals (Fig. 8, channel III) and 2S albumin (Fig. 8,channel I) were replaced by bands in less than 10 KD. Thisindicated that their macromolecular proteins were enzymatichydrolyzed by free protease. The same results happened when theimmobilized protease was used. 2S albumin (Fig. 8, channel VIII)was hydrolyzed by immobilized protease and rapeseed mealstreated by immobilized protease obtained immediately (Fig. 8,channel VII) or kept for 20 days (Fig. 8, channel V), whosemacromolecular proteins were not able to be detected. All theresults revealed that the macromolecular proteins of rapeseedmeals are hydrolyzed by alkaline protease into the mall molecules

Fig. 8. The electrophoretograms of hydrolysate of 2S albumin and rapeseed meals

hydrolyzed by immobilized protease. (From left to right: I: 2S albumin hydrolyzed

by free protease; II: 2S albumin; III: rapeseed meals hydrolyzed by free protease;

IV: rapeseed meals; Marker; V: rapeseed meals hydrolyzed by immobilized

protease (immobilized kept at 4 1C for 20 days, 50 mg rapeseed meals kept at 80 1C

for 30 min to dissolve completely); VI: rapeseed meals hydrolyzed by fresh

immobilized protease (50 mg rapeseed meals were ultrasonically agitated to

dissolve completely); VII: rapeseed meals hydrolyzed by fresh immobilized

protease (50 mg rapeseed meals kept at 80 1C for 30 min to dissolve completely);

VIII: 2S albumin hydrolyzed by immobilized protease).

Fig. 7. The storage stability of immobilized (a) and free (b) protease.

such as polypeptides or amino acids. Immobilized protease hadrather high catalytic activity.

The degree of hydrolysis is the percentage of broken peptidebonds by enzymatic hydrolysis reaction with physical andchemical methods to the total amount of peptide bonds ofprotein. It is used to measure the extent that the protein ishydrolyzed into free amino acids and verify the effect of enzymecatalysis. In brief, the degree of hydrolysis is an importantparameter to measure the catalytic activity of enzyme. Thedegree of hydrolysis of immobilized protease catalyzing rapeseedmeals could reach 9.86%, and the free protease can reach 10.41%.It could be seen that immobilized protease retained the sameability to hydrolyze rapeseed meals. On the other hand, a lot ofother factors affect the degree of hydrolysis such as the scale ofprotease and substrate and reaction time. Therefore, the catalyticcapability of immobilized protease may be improved after furtheroptimization. But the work here can show this magneticnanoparticles based enzyme carrier is a promising approach towiden the immobilized enzyme applications.

4. Conclusions

In this work, immobilized protease was prepared by immobi-lizing alkaline protease onto the surface of amino silane coatedmagnetic nanoparticles. All the results demonstrated that theimmobilized protease presented good catalytic activity, and canbe separated easily by applying external magnetic field, providinga feasible way to produce active peptides continuously.

Acknowledgements

The work was supported by the National High TechnologyResearch and Development Program of China (863 Program:2007AA10Z328, 2007AA100703), the National Natural ScienceFoundation of China (Grant no. 30670553). We also thankAnalytical and Testing Center (HUST) for the help of measure-ment. This work was also supported by the Graduate InnovationFund of Huazhong University of Science and Technology(HF05222007170).

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