酶催化柚皮苷水解

Applied Surface Science 257 (2011) 4096–4099

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Applied Surface

Science

j o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /a p s u s

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Immobilization of naringinase on mesoporous molecular sieve MCM-41and its application to debittering of white grapefruit

Shengjiao Lei,Yongxia Xu,Gang Fan,Ming Xiao,Siyi Pan ?

College of Food Science and Technology,Huazhong Agricultural University,Wuhan 430070,China

a r t i c l e i n f o Article history:

Received 7September 2010

Received in revised form 1December 2010Accepted 1December 2010

Available online 9 December 2010Keywords:Naringinase Immobilization MCM-41Debittering

a b s t r a c t

Naringinase was bound to mesoporous silica MCM-41via adsorption with glutaraldehyde and used to debitter white grapefruit.K m value of the immobilized naringinase was lower than that of free naring-inase.The immobilized catalysts showed excellent thermal stability and storage stability and could be recycled 6times retained about 44.57%activities.The unaltered structural order of the prepared cata-lyst was characterized with reference to bulky and surface properties by infrared spectroscopy (FT-IR),elemental analysis and nitrogen adsorption–desorption isotherms analysis.

Published by Elsevier B.V.

1.Introduction

Naringin is one of the major bitter substances in grapefruit juice.Naringinase,an enzymatic complex with ?-l -rhamnosidase (EC 3.2.1.40)and ?-d -glucosidase (EC 3.2.1.21)activities [1],can hydrolyze naringin to rhamnose,glucose and naringenin [2],which are not bitter.To reduce naringin in grape fruit,naringinase has been immobilized on various matrices using several methods including adsorption,cross linking,adsorption followed by cross linking,covalent attachment and physical entrapment [3–9].

Mesoporous molecular sieve has tunable pore size,large sur-face area,pore volume,high adsorption capacity,and an ordered porous network for free diffusion of substrates and reaction prod-ucts [10].Those unique properties make it a potentially ideal matrix for enzyme immobilization.Reports [11,12]have appeared on the catalytic application of MCM-41as a catalyst support on the immobilization of a couple of enzymes and it showed good performance.

In the present study,naringinase was adsorbed onto MCM-41and crosslinked to silylated MCM-41by glutaraldehyde which provided a large number of aldehyde groups for the immobi-lized enzyme.Hydrolytic activity,thermal stability,reusability and kinetic mechanism of the immobilized naringinase were investigated and compared to the free one.Finally,immobilized naringinase was applied to hydrolyze naringin in the white grapefruits.

?Corresponding author.

E-mail address:pansy 111@https://www.360docs.net/doc/6016408924.html, (S.Pan). 2.Materials and methods 2.1.Materials

Naringinase (CAS Number 9068-31-9)from Penicillum decum-bens (activity:396U g ?1solid)and naringin (96.6%)were obtained from Sigma Chemical Co (St Louis,MO,USA).Naringenin (HPLC,98%)and limonin (HPLC,98%)were from Chengdu Pus Bio-technology Co.,Ltd.Mesoporous MCM-41was from Shanghai Novel Chemical Technology Co.,Ltd.Acetonitrile and methanols were HPLC grade,and all other chemicals were analytical grade and obtained from various sources.White grapefruits used in this study were obtained from the orange garden of Wangchunhua Syrup Co.Ltd.,Songzi city,Hubei Province.

2.2.Preparation of immobilized naringinase by mesoporous MCM-41

8.0mg of naringinase in 20mL acetate buffer (0.2M,pH 4.0)was mixed with 0.2g of MCM-41(1.0%)and 0.4g glu-taraldehyde (2.0%)in a conical ?ask by stirring at 15?C and 170rpm for 6h [13].After 6h adsorption,MCM-41-naringinase was separated by centrifugation at 15?C and 10,000rpm (cov-erse to 0.2g)for 10min.The pelleted MCM-41-naringinase was washed three times with 0.2M acetate buffer (pH 4.0)and dried under room temperature for 3h and stored at 4?C before used [14].

0169-4332/$–see front matter.Published by Elsevier B.V.doi:

10.1016/j.apsusc.2010.12.003

S.Lei et al./Applied Surface Science257 (2011) 4096–40994097

2.3.Assay of the free and immobilized naringinase activity

The activities of the free and immobilized naringinase were measured by analyzing residual naringin using a modi?cation of Davis’method[15].1.0mL reaction mixture(0.2M acetate buffer, pH4.0)containing0.3mg naringin and0.1mg free or equivalent immobilized naringinase was incubated and shaked at60?C and 150rpm for30min.After the reaction,4.9mL of90%diethyleneg-lycol(DEG)was added immediately.Then,0.1mL NaOH(4.0M) and4mL distilled water were added to the fully blended mixture. The assay mixture was allowed to stand at40?C for10min and the intensity of the developed yellow color was determined spec-trophotometrically at420nm.One unit(U)of activity was de?ned as the amount of free or immobilized enzyme required to hydrolyze 1?g naringin per min under the above assay conditions.Relative activity is expressed taken as100%the maximum activity in each case.

2.4.Stabilities of naringinase and MCM41–naringinase

To study the thermal stabilities of the free and immobilized naringinase,both enzymes were incubated at60?C and sampled at different incubation time points.The operational stability of the MCM41–naringinase was determined by repeatedly using in the hydrolysis of naringin.After each reaction run,the mixture was cen-trifuged and decanted.The regenerated MCM41–naringinase was washed with0.2M acetate buffer(pH4.0)to remove any residual substrate within the molecular matrix.The free naringinase was stored in0.2M acetate buffer(pH4.0)at4?C and the MCM41–naringinase was stored at?18?C and4?C,respectively.The stor-age stabilities of free and immobilized naringinase were measured by determining their activities at a certain intervals during stor-age.

2.5.Characterizations of MCM-41and MCM41–naringinase

The molecular sieve MCM-41and MCM41–naringinase matrix were characterized by Fourier transform-infrared spectrophotometer(FT-IR),elemental analysis and the nitro-gen adsorption–desorption isotherms.FT-IR in the range of 4000–400cm?1was received from a Perkin Spectrum using KBr wafers(sample0.05wt%).The C,H,N contents were determined by CHN elemental analyses(VARIO EL III,Elementar).Other elements in the samples were measured by X-ray?uorescence(XRF) analyses(S4PIONEER,AXS).The nitrogen adsorption–desorption data were recorded at liquid nitrogen temperature77.3K using

a Quantachrome Autosorb-1MP-CR.Before measurement,about

0.05g of sample was degassed at200?C for3h.The speci?c surface areas of the samples were calculated by the multiple-point Brunauer–Emmett–Teller(BET)method using the adsorption data ranging from p/p0=0.05–0.35.The pore diameter distribution curves were computed by using the Barret–Joyner–Halenda(BJH) method,and pore sizes were obtained from the peak positions of the distribution curves.

2.6.Debittered white grapefruit by free naringinase and

MCM41–naringinase

The white grapefruits were cut into halves,squeezed,and then the juice was centrifuged at15?C and4000rpm(100mL)for 15min.The clear juices were incubated with moderate naringi-nase or MCM41–naringinase at60?C for30min.After treatment, the reaction was stopped in boiling water for5min.The naringin and produced naringenin were measured with HPLC(Waters,USA) according to Ribeiro and Ribeiro[16]

.

Fig.1.Lineweaver–Burk plots for free and immobilized naringinase.

3.Results and discussions

3.1.Kinetic parameters for naringinase and MCM41–naringinase

The kinetic mechanism of free and MCM41–naringinase, the Lineweaver–Burk plot,is shown in Fig.1.For both free and MCM41–naringinase,the average correlation coef?cients of the plots https://www.360docs.net/doc/6016408924.html,pared with free enzyme,the MCM41–naringinase exhibited lower V max and K m values.The result was consistent with Mishra and Kar[17]and Puri et al.’s [18]studies,but was inconsistent with that of Busto et al.[19]. The lower K m value of the immobilized enzyme indicated that the matrix structure caused less steric limitations and the substrate was free to interact with immobilized enzyme by covalent binding.This was due to either enzyme immobilized in mesoporous molecular sieve with multiple point attachment or with higher hydrophobic-ity exhibits,which results in a more accessibility of the substrate to the active sites of the immobilized enzyme.

3.2.Stability of naringinase and MCM41–naringinase

Thermal stability of enzyme is one of the most important cri-teria for long term and commercial application.Fig.2showed the relative residual activities of naringinase and MCM41–naringinase after different incubation time points.MCM41–naringinase was more stable than free naringinase after180min of incubation.The signi?cant thermal stability of the immobilized naringinase could be resulted from the inside porous structure and microenviron-ment of the supports which prevented some intermolecular or protein-surface interactions[20].This also probably meant that the structure of immobilized enzymes site was strengthened,which created strong bonds between the macromolecule and the carrier.

The results of the operational stability of MCM41–naringi-nase were shown in Table1.The activity of MCM41–naringinase retained about44.57%after reusing for6times.It demonstrated that the immobilized naringinase had a good reusability,which made it useful for potential commercial applications.MCM41–naringinase can be reused several times,which is an important advantage compared to the free enzyme.

Enzymes are not stable during storage in solutions and their activities decreased gradually with the time.Hence,it is important to examine the storage stability for the application of immobilized enzyme systems in the physiological environment.Fig.3showed

4098S.Lei et al./Applied Surface Science

257 (2011) 4096–4099

Fig.2.The thermal stability of free and immobilized naringinase.

Table 1

The operational stability of MCM41–naringinase.Cycle no.Relative activity (%)1100

292.14±2.72378.55±1.63464.82±0.47554.76±0.786

44.57±0.40

the relative activities of free naringinase and MCM41–naringinase at different storage times and different temperatures.The frozen MCM41–naringinase retained 81%of its activity while the unfrozen one only remained 42%at day 30.When stored at 4?C for 6days,the immobilized naringinase activity was retained 90%whereas the activity of free naringinase decreased to 80%.After 15days,the free enzyme was deactivated considerably whereas MCM41–naringinase retained 86%of its activity up to 30

days.

Fig.3.The storage stability of free and immobilized naringinase.

400

80012001600

200024002800320036004000

05

10

15

20

25

A U

Wavenumber (cm -1

)

(a) MCM-41

(b) MCM41-Naringinase

Fig.4.FT-IR spectrum of (a)MCM-41and (b)MCM41–naringinase support materi-als.

The results clearly suggested that naringinase immobilized on mesoporous molecular sieve MCM-41had higher stability than free enzyme,which lost most of its initial activity around 15days.To day,the mechanisms underlying the storage stability of immobilized enzyme are complex and still unclear.The ability of mesoporous materials of keeping the active conformation of enzyme was postulated to be a critical factor.3.3.Characterization of MCM41–naringinase

Fig.4showed the FT-IR spectra of MCM-41and MCM41–naringinase support materials.Both infrared spectra exhibited the major absorption bands associated with network Si–O–Si vibrational modes at 460,1072and 1300cm ?1,along with Si–OH asymmetric stretching at 955cm ?1[21].A very broad absorption envelope at 3460cm ?1was attributed to hydrogen-bonded SiO–H stretching vibration of physically bound water while stretching of the C–Si bond n (C–Si)was located at 780cm ?1.The peak at 1634cm ?1was attributed to the vibration of adsorbed water and the others were from the frame symmetric and asym-metric ?exible vibrations of Si groups [22].The similarities of the two spectra indicated that MCM-41structure did not change greatly after crosslinked with the enzyme.

Fig.5is the nitrogen adsorption–desorption isotherms of the samples.Obviously,all the samples exhibited type IV isotherms with type H1hysteresis according to the IUPAC classi?cation [23],indicating that all samples were typical of ordered mesopores.The free surface area obtained from BET adsorption isotherms was 859m 2/g for MCM-41alone,and the area for MCM41–naringinase was found to be 782m 2/g.A comparison of adsorption isotherms for MCM-41before and after enzyme immobilization showed that the pore size did not change signi?cantly,which is due to a small amount of immobilized enzyme,a signi?cant portion of which could be attached to the external surface and/or located at the pore openings (the latter case could result in a partial blocking of pores).The BJH pore size analysis showed that the pore size of MCM-41was probably about 3.4nm.In comparison,the pore size of MCM41–naringinase was smaller than that of MCM-41.A possible

Table 2

Elemental analyses of free naringinase,MCM-41and MCM41–naringinase.Material

C (%)H (%)N (%)Si (%)O (%)Free naringinase 40.8507.031 4.590–

–

MCM -41

0.616 2.918–

42.63853.728MCM41–naringinase

1.387

4.474

0.260

41.636

52.143

S.Lei et al./Applied Surface Science257 (2011) 4096–40994099

Table3

Naringin hydrolysis in white grapefruit by free and immobilized naringinase.

Grapefruit Naringin

conversion(%)Naringenin

formation(mg/L)

Prulin formation

(mg/L)

Linmonin

(mg/L)

Free naringinase96.0991.9933.2716.53

MCM41–naringinase95.0369.0960.77

14.49

Fig. 5.Nitrogen adsorption–desorption isotherms of MCM-41and MCM41–naringinase support materials.

explanation is lack of change in the pore size due to the attachment of enzyme.Also,it should be noted that the classical BJH method underestimates the pore size of small mesopores.

The elemental analyses data of free naringinase,MCM-41and MCM41–naringinase were given in Table2.The physically adsorbed water was removed from samples prior to elemental analysis by drying them under reduced pressure[24].Therefore,the given hydrogen analysis in Table2included the hydrogen of the surface hydroxyls of silica and also occluded water hydrogen.The nitrogen and hydrogen contents in the MCM41–naringinase were roughly increased while no nitrogen was detected in MCM-41silica sample. As expected,no apparent change was observed in main elements of the matrix.The results indicated that naringinase was successfully immobilized on the MCM-41.

All of the above data indicated that naringinase was bound to MCM-41while the matrix still maintained the typical uniform mesopores structure.

3.4.Debittered white grapefruit by the free and

MCM41–naringinase

The decrease in naringin content can be directly correlated with reduction in bitterness.From the amount of residual naringin present,the percentage reduction in bitterness was evaluated.The results were showed in Table3.It was observed that naringin of 96.09%and95.03%were convered into prulin and naringenin by the free and MCM41–naringinase,respectively.More importantly, the experiments data also indicated that the debittering ef?ciency of free and MCM41–naringinase to juice was quite different,which determined by the formation of naringenin and prulin.The concen-tration of naringenin produced with free naringinase was almost more three times than that of the prulin,while their amounts-produced with MCM41–naringinase were almost edentical.Prunin bitterness is one-third of that of naringin,and naringenin is almost tasteless as described in Chien’s work[2].According to Soares and Hotchkiss’s[25]studies,some bitterness in grapefruit juice is acceptable to consumers,since it contributes to the characteristic taste and?avor.

4.Conclusions

This immobilized preparation of naringinase seemed to be suit-able for industrial use,as the high thermal stability and storage stability may make it useful for debitterring of grapefruit.The cova-lent enzyme immobilization process generates an insoluble and quite stable form of the enzyme,whilst retaining catalytic prop-erties,and allows enzymes to work in a wide range of temperature. The pore size distribution curves showed a high amount of naring-inase loading in MCM-41.Reusability of MCM41–naringinase on naringin hydrolysis showed about44.57%of hydrolysis up to6runs and naringin conversion on the juice up to95%.All these results suggest that the mesoporous sieve MCM-41is an ef?cient carrier material for higher amount of naringinase immobilization.Thus the MCM41–naringinase can be considered as a potential biocatalyst for the biotechnological and industrial applications.

Acknowledgements

We thank Mr.Wang Kexing for?nancial support on this study and Wangchunhua Syrup Co.,Ltd.for providing the white grape-fruits.We also thank Ms.Qin Wenmin(Ames,USA)for revising the manuscript.

References

[1]F.Soria1,G.Ellenrieder,M.Grasselli,A.A.Navarro del Canizo,O.Cascone,

Biotechnol.Lett.26(2004)1265.

[2]P.Chien,F.Sheu,Y.Shyu,J.Food Drug Anal.9(2001)115.

[3]A.C.Olson,G.M.Gray,D.G.Guadagni,J.Food Sci.44(1979)1358.

[4]G.Spagna,R.N.Barbagallo,D.Casarini,P.G.Pifferi,Enzyme Microb.Technol.28

(2001)427.

[5]M.Puri,M.Seth,S.S.Marwaha,R.M.Kothari,Food Biotechnol.15(2001)13.

[6]S.Yadav,K.Yadav,Ind.J.Chem.Technol.8(2001)314.

[7]G.Sekeroglu,S.Fad?loglu,F.Gogus,Eur.Food Res.Technol.224(2006)55.

[8]J.Russo,M.I.Barreiro,H.Vila-Real,H.Maria,J.Biotechnol.131(2007)S74.

[9]H.Birgisson,J.O.Wheat,O.Gudmundur,Enzyme Microb.Technol.40(2007)

1181.

[10]S.Hudson,J.Cooney,E.Magner,Angew.Chem.Int.Ed.47(2008)8582.

[11]H.H.P.Yiu,P.A.Wright,N.P.Botting,Micropor.Mesopor.Mater.44–45(2001)

763.

[12]J.Choma,M.Jaroniec,Appl.Surf.Sci.253(2007)5587.

[13]I.Migneault,C.Dartiguenave,M.J.Bertrand,K.C.Waldron,Biotechniques37

(2004)798.

[14]Y.Wu,Y.J.Wang,G.S.Luo,Y.Y.Dai,Bioresource Technol.101(2010)841.

[15]W.Davis,Anal.Chem.19(1947)476.

[16]I.A.Ribeiro,M.H.L.Ribeiro,Food Control19(2008)432.

[17]P.Mishra,R.Kar,J.Food Sci.68(2003)1229.

[18]M.Puri,K.Harsimran,J.F.Kennedy,Chem.Technol.Biotechnol.80

(2005)1160.

[19]M.D.Busto,V.Meza,N.Ortega,M.Perez-Mateos,Food Chem.104(2007)1177.

[20]K.Ogawa,B.Wang,E.Kokufuta,Langmuir17(2001)4704.

[21]Y.Wei,H.Dong,J.G.Xu,Q.W.Feng,Chem.Phys.Chem.9(2002)802.

[22]Q.R.Peng,Y.Yang,Y.Z.Yuan,J.Mol.Catal.A:Chem.219(2004)175.

[23]K.S.W.Sing,D.H.Everett,R.A.W.Haul,L.Moscou,R.A.Pierotti,J.Rouquerol,T.

Siemieniewska,Pure.Appl.Chem.57(1985)603.

[24]M.Pirouzmand,M.M.Amini,N.Safari,J.Colloid Interface Sci.319(2008)199.

[25]N.Soares,J.Hotchkiss,J.Food Sci.63(1998)61.