Il

Ja

b

a

ARR1AA

KALIAKS

1

mpeoraita

st

(

0d

Colloids and Surfaces B: Biointerfaces 77 (2010) 166–173

Contents lists available at ScienceDirect

Colloids and Surfaces B: Biointerfaces

journa l homepage: www.e lsev ier .com/ locate /co lsur fb

mmobilization of acid phosphatase on uncalcined and calcined Mg/Al-CO3

ayered double hydroxides

un Zhua,b, Qiaoyun Huanga,∗, Massimo Pignab, Antonio Violanteb,∗∗

Key Laboratory of Subtropical Agriculture and Environment, Ministry of Agriculture, College of Resources and Environment, Huazhong Agricultural University, Wuhan 430070, ChinaDepartment of Soil, Plant, Environment and Animal Production Science, Faculty of Agriculture, University of Naples “Federico II”, Via University 100, 80055 Portici (Naples), Italy

r t i c l e i n f o

rticle history:eceived 25 November 2009eceived in revised form8 December 2009ccepted 25 January 2010vailable online 4 February 2010

eywords:cid phosphataseayered double hydroxidesmmobilizationctivityinetics

a b s t r a c t

Acid phosphatase was immobilized on layered double hydroxides of uncalcined- and calcined-Mg/Al-CO3 (Unc-LDH-CO3, C-LDH-CO3) by the means of direct adsorption. Optimal pH and temperature for theactivity of free and immobilized enzyme were exhibited at pH 5.5 and 37 ◦C. The Michaelis constant (Km)for free enzyme was 1.09 mmol mL−1 while that for immobilized enzyme on Unc-LDH-CO3 and C-LDH-CO3 was increased to 1.22 and 1.19 mmol mL−1, respectively, indicating the decreased affinity of substratefor immobilized enzymes. The residual activity of immobilized enzyme on Unc-LDH-CO3 and C-LDH-CO3

at optimal pH and temperature was 80% and 88%, respectively, suggesting that only little activity was lostduring immobilization. The deactivation energy (Ed) for free and immobilized enzyme on Unc-LDH-CO3

and C-LDH-CO3 was 65.44, 35.24 and 40.66 kJ mol−1, respectively, indicating the improving of thermalstability of acid phosphatase after the immobilization on LDH-CO3 especially the uncalcined form. Bothchemical assays and isothermal titration calorimetry (ITC) observations implied that hydrolytic stability

tabilityof acid phosphatase was promoted significantly after the immobilization on LDH-CO3 especially thecalcined form. Reusability investigation showed that more than 60% of the initial activity was remainedafter six reuses of immobilized enzyme on Unc-LDH-CO3 and C-LDH-CO3. A half-life (t1/2) of 10 days wascalculated for free enzyme, 55 and 79 days for the immobilized enzyme on Unc-LDH-CO3 and C-LDH-CO3

when stored at 4 ◦C. Therefore, immobilization of acid phosphatase on Unc-LDH-CO3 and C-LDH-CO3 byfectiv

and

direct adsorption is an efin agricultural production

. Introduction

Acid phosphatase, which comes from microorganisms, ani-als, and plants, catalyzes the hydrolysis of a wide range of

hosphomonoesters and phosphoproteins nonspecifically in acidicnvironments [1,2]. It plays a key role in the transformation ofrganic phosphorus to orthophosphate, the only type of phospho-us source taken up by crops [3,4]. Acid phosphatase is also used asn indicator enzyme for monitoring the risk of various pollutantsn soils [5–7]. Moreover, the enzyme is important in promoting

he degradation of organophosphorous pesticides in terrestrial andqueous environments [8,9].

It has been reported that free enzymes generally have ahort-lived activity because of rapid denaturation and degrada-ion [10–12]. Immobilization on ideal support is an effective way

∗ Corresponding author. Tel.: +86 27 87671033; fax: +86 27 87280670.∗∗ Corresponding author. Tel: +39 081 2539175; fax: +39 081 2539186.

E-mail addresses: qyhuang@mail.hzau.edu.cn (Q. Huang), violante@unina.itA. Violante).

927-7765/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.colsurfb.2010.01.020

e means and would have promising potential for the practical applicationenvironmental remediation.

© 2010 Elsevier B.V. All rights reserved.

to reuse or for improving their stability [13–16]. Ohmiya et al.[17] have immobilized acid phosphatase on porous glass beadsfor the dephosphorylation of casein. Kurita et al. [18] have com-pared the immobilization of acid phosphatase on 2-mercapto- and6-mercapto-chitin, 6-mercapto-chitin was found to be a better sup-port according to the durability of immobilized enzyme. Chang andJuang [13] have taken composite beads of chitosan and activatedclay as support for the immobilization of acid phosphatase, theimmobilized enzyme kept 90% of its original activity after 50 timesof reuse. The immobilization of acid phosphatase on wet/driedpure chitosan beads, wet/dried chitosan–ZnO2 composite beadshas also been observed by Chang and Juang [14], chitosan–ZnO2composite as wet form was suggested as the most promising sup-port for immobilization application. Moreover, acid phosphatasehas been immobilized on montmorillonite and chitosan by Lai andShin [19] to enhance the content of phosphorus in soils. However,most of these reported carriers are organic or semi-organic matri-

ces and would subject to microbial attack in practical application.In order to find more effective materials as immobilizing agents,we have carried out an investigation on the immobilization of acidphosphatase by a type of inorganic clay, namely, layered doublehydroxides (LDHs).

es B: B

[taTci[silas[brtpho(ctaN(r

obkipp

2

2

w

2L

bLCs

stawM

oa(Lt

2

w

J. Zhu et al. / Colloids and Surfac

LDHs have a general formula of [M1−x2+ Mx

3+ (OH)2]x+

Ax/nn−·mH2O]x−, where M2+, M3+ and An− are divalent cation,

rivalent cation and interlayer anion, respectively, x is defineds M2+/(M2+ + M3+) and varies between 0.20 and 0.33 [20,21].hese so-called anionic clays, which exist in nature or easily andheaply synthesized under laboratory conditions, consist of pos-tively charged layers and negatively charged interlayer anions20,22]. The positive charges are developed due to the partial sub-titution of divalent metal ions by trivalent cations and balanced bynterlayer anions [20,23]. By calcination above 450 ◦C, LDHs wouldose their layer structure due to dehydroxylation and decarbon-tion and form highly active composite metal oxides with largerurface area, higher porosity and greater anion exchange capacity21,24]. Over the past decades, uncalcined- and calcined-LDHs haveeen used in many fields such as pharmaceutics and environmentalemediation [25,26]. They have also been found to be available ashe host material for enzymes due to their charged structure andorous surface. Ren et al. [27] reported that calcined-Mg/Al-CO3ave higher affinity for penicillin G acylase but lower percentagef residual activity than calcined-Zn/Al-CO3. Alkaline phosphataseAIP) immobilized within a Mg2Al-LDH by “soft chemistry” copre-ipitation synthesis showed 36–44% of residual activity [28]. Thehermal and storage stability of lipase was increased obviouslyfter the immobilization on layered double hydroxide of Zn/Al-O3, Ni/Al-NO3, Mg/Al-NO3 and Mg/Al-sodium dodecyl sulphate

SDS) by direct adsorption [15,16], which is a physical method andelatively easier and cheaper compare to chemical method [15].

The objectives of the present study are to investigate theptimal conditions for the immobilization of acid phosphatasey direct adsorption on Unc-LDH-CO3 and C-LDH-CO3 and theinetic property, thermal, hydrolytic, reusable and storage stabil-ty of the immobilized enzyme. The potentials of immobilized acidhosphatase in the regulation of soil fertility and remediation ofesticides pollution might be evaluated.

. Materials and methods

.1. Enzyme

Acid phosphatase (EC3.1.3.2 Type II, 1.0 units mg−1 from potato)as purchased from Sigma Chemical Company.

.2. Synthesis and characterization of uncalcined and calcinedDH-CO3

The layered double hydroxide of Unc-LDH-CO3 was synthesizedy coprecipitation as described by Violante et al. [29]. Part of Unc-DH-CO3 was calcined at 500 ◦C in an oven for 4 h. Both Unc-LDH-O3 and C-LDH-CO3 were used for the following immobilizationtudies.

The surface area of the sample was determined by highpeed automated surface area and pore size analyzer (Quan-achrome Autosorb-1, USA), it was 90.5 m2 g−1 for Unc-LDH-CO3nd 176.8 m2 g−1 for C-LDH-CO3. The point of zero charge (PZC)as 7.9 for Unc-LDH-CO3 and 8.7 for C-LDH-CO3 as measured byehlich method [30].The SEM images of freeze dried LDHs–enzyme complexes were

btained by a JSM-6390 scanning electron microscope (JEOL, Japan)fter the gold coating in vacuum by a JFC-1600 sputter coaterJEOL, Japan). The X-ray diffraction (XRD) patterns of LDH-CO3 andDHs–enzyme complexes were obtained by a Rigaku diffractome-er (Rigaku Corporation, Japan) equipped with Cu K� radiation.

.3. Immobilization of acid phosphatase

In a 10 mL centrifuge tube, ten milligram of LDHs were mixedith 4 mL of 10 mmol L−1 acetate buffer (pH 5.5) containing 200,

iointerfaces 77 (2010) 166–173 167

400, 600, 800, 1000, 1200, 1400, and 1600 �g of acid phosphatase.The mixture was shaken at 25 ◦C and 250 rpm for 2 h and then cen-trifuged at 10,000 × g for 20 min. The supernatant was collectedand the residue was washed twice by 1.5 mL of acetate buffer.After centrifugation again, the washings were collected and theenzyme on the final residue (LDHs–enzyme complex) was theimmobilized acid phosphatase. The concentration of acid phos-phatase in the first supernatant and the following washings wasdetermined at 280 nm by UV-spectrophotometer using BSA as thestandard. The amount of acid phosphatase immobilized was cal-culated by subtracting the quantity of enzyme in solution fromenzyme added initially. The immobilization of acid phosphatasewas also investigated as a function of residence time and bufferpH. The immobilized acid phosphatase which was prepared bythe immobilization of the enzyme with the initial concentrationof 250 �g mL−1 in 4 mL acetate buffer on 10 mg LDHs at pH 5.5 and25 ◦C was employed for the following activity and stability assays.

2.4. Activity of free and immobilized acid phosphatase

The activity of free and immobilized acid phosphatase weredetermined as follows:

The pH-activity profiles were constructed by re-suspending theLDHs–enzyme complex by 2 mL of 10 mmol L−1 buffer at variouspHs (acetate buffer for pH 4.5, 5.0, 5.5, 6.0, 6.5 and Tris–HCl bufferfor pH 7.0). One hundred microlitres of free acid phosphatase(0.5 mg mL−1) or the thoroughly re-suspended LDHs–enzymecomplex was then mixed with 900 �L of corresponding buffer con-taining 8 �mol �-nitrophenyl phosphate (pNPP). After incubationat 37 ◦C for 1 h, the enzymatic reaction was terminated immedi-ately by the addition of 1 mL of 1 mol L−1 NaOH solution [12]. Theconcentration of enzymatic product �-nitrophenol was determineddirectly at 405 nm spectrophotometrically. The specific activity offree and immobilized acid phosphatase was defined as �mol �-nitrophenol released by the catalysis of 1 mg acid phosphatasewithin 1 min.

The temperature–activity profiles were constructed by conduct-ing the enzymatic reaction in 10 mmol L−1 of acetate buffer (pH 5.5)with 8 �mol pNPP at 15, 25, 37, 45, 55, and 65 ◦C for 1 h.

The catalytic kinetics was analyzed by incubating the enzymein 10 mmol L−1 of acetate buffer (pH 5.5) containing 1, 2, 4, 6, and8 �mol pNPP at 37 ◦C for 1 h.

2.5. Stability of free and immobilized acid phosphatase

The thermal stability was investigated by incubating 100 �Lof free enzyme (0.5 mg mL−1) or the thoroughly re-suspendedLDHs–enzyme complex in the absence of substrate at 50, 60, and70 ◦C for 30, 60, 90, 120, and 150 min. The activity was then deter-mined after the further incubation with 900 �L of 10 mmol L−1 ofacetate buffer (pH 5.5) containing 8 �mol pNPP at 37 ◦C for 1 h.

The hydrolytic stability was estimated by mixing 100 �Lof free enzyme (0.5 mg mL−1) or the thoroughly re-suspendedLDHs–enzyme complex with 900 �L of 10 mmol L−1 of acetatebuffer (pH 5.5) containing 25 �g of proteinase K. The mixture wasthoroughly dispersed and then incubated at 37 ◦C for 1 h, 2 h and24 h, respectively. One millilitre of 10 mmol L−1 of acetate buffer(pH 5.5) containing 8 �mol pNPP was then added immediatelyinto the tube and the mixture was incubated in succession at37 ◦C for 1 h. No proteinase exposure was tested as the controlfor free and immobilized enzyme. The hydrolytic stability of free

and immobilized phosphatase was calculated according to the fol-lowing expression: hydrolytic stability (%) = (specific activity of acidphosphatase in the presence of proteinase/specific activity of acidphosphatase in the absence of proteinase) × 100. The hydrolyticstability of free and immobilized enzyme was also investigated by

168 J. Zhu et al. / Colloids and Surfaces B: Biointerfaces 77 (2010) 166–173

sing e

IAdp

c

b5t

3

3

lc

Fig. 1. Immobilization of acid phosphatase on LDHs with increa

TC with an isothermal microcalorimeter TAM III (ThermometricB, Sweden). Net hydrolytic heat was calculated by deducting theilution and/or adsorption heat of proteinase (sample without acidhosphatase) from the total heat (sample with acid phosphatase).

For reusability, both immobilized enzyme were employed toatalyze the hydrolysis of pNPP repeatedly in a continuous cycles.

The storage stability was observed by storing the free and immo-ilized enzymes in 10 mmol L−1 of acetate buffer (pH 5.5) at 4 ◦C for, 10, 30, 60, and 90 days. The residual activity was determined inime by the method described above.

. Results

.1. Immobilization of acid phosphatase on LDHs

Figure 1 depicts the amount of acid phosphatase immobi-ized by Unc-LDH-CO3 and C-LDH-CO3 with increasing enzymeoncentrations, reaction time and buffer pH. The immobilization

Fig. 2. SEM micrographs of Unc-LDH-CO3 (A) and C

nzyme concentrations (A), reaction time (B) and buffer pH (C).

of the enzyme on both supports reached a plateau value at anenzyme concentration of 250 �g mL−1 within 2 h. The maximumimmobilization was observed at pH 5.5. The percentage of acidphosphatase immobilized at optimal conditions by Unc-LDH-CO3and C-LDH-CO3 was 59% and 73%, respectively, indicating higherimmobilization of acid phosphatase on the latter support than thaton the former one.

3.2. Characterization of LDHs and LDHs–acid phosphatasecomplex

The LDHs and/or LDHs–enzyme complexes were characterizedby SEM and XRD techniques. A flocky morphology of C-LDH-CO3

and a studded shape of Unc-LDH-CO3 after the immobilization ofacid phosphatase were recorded by SEM (Fig. 2). The XRD patternsof Unc-LDH-CO3 in Fig. 3 showed some typical peaks which char-acterized the widely reported hydrotalcite [29], a basal space of0.756 nm was calculated from the 003 reflection. For C-LDH-CO3,

-LDH-CO3 (B) after enzyme immobilization.

J. Zhu et al. / Colloids and Surfaces B: Biointerfaces 77 (2010) 166–173 169

Fe

tpbpc(cote

3

aartcoa6fcb

pfpei

Table 1Kinetic parameters of free (F-E) and immobilized acid phosphatase on LDHs (I-E onUnc-LDH-CO3 and I-E on C-LDH-CO3) by Michaelis–Menten equation.

Enzyme Km (mmol mL−1) Vmax (�mol mg−1 min−1)

ig. 3. Powder XRD patterns of Unc-LDH-CO3 and C-LDH-CO3 before and afternzyme immobilization.

he typical peaks of hydrotalcite disappeared while new broadeaks appeared (Fig. 3), indicating the collapse of layered dou-le structure and the formation of Mg–Al mixed oxides. The XRDatterns of both Unc-LDH-CO3–enzyme and C-LDH-CO3–enzymeomplex were similar to that of the corresponding pure mineralFig. 3), suggesting that acid phosphatase was limited to be inter-alated into Unc-LDH-CO3 and the immobilization of the enzymen the surface of C-LDH-CO3 prevented its structural reconstruc-ion. Therefore, acid phosphatase was immobilized mainly on thexternal surface of Unc-LDH-CO3 and C-LDH-CO3.

.3. Activity of free and immobilized acid phosphatase

As revealed by the pH-activity profiles (Fig. 4A), the maximumctivity for free and immobilized acid phosphatase were observedt pH 5.5, where the enzyme on Unc-LDH-CO3 and C-LDH-CO3etained 80 and 88% of the residual activity. These results indicatedhat the optimal pH for the activity of acid phosphatase did nothange after the immobilization. The optimal pH for the activityf acid phosphatase was also reported at pH 5.5 and kept constantfter the immobilization on Red soil colloids, kaolinite, goethite and-mercapto-chitin [12,18]. However, a shift of optimal pH from 5.2or free acid phosphatase to 2.9 for the immobilized enzyme onomposite beads of chitosan and ZrO2 powders was documentedy Chang and Juang [13].

Figure 4B presents the activity of free and immobilized acid◦ ◦

hosphatase at temperatures from 15 C to 60 C. The activity of

ree and immobilized enzyme increased with the increase of tem-erature from 15 ◦C to 37 ◦C and reached maximum at 37 ◦C. Freenzyme lost its activity rapidly above 37 ◦C, whereas enzymesmmobilized on both supports were more stable. The activity of

Fig. 4. Activity of free (F-E) and immobilized acid phosphatase on LDHs (I-E on Un

F-E 1.09 0.68I-E on Unc-LDH-CO3 1.22 0.57I-E on C-LDH-CO3 1.19 0.62

acid phosphatase immobilized on LDH-CO3 exceeded that of freeenzyme at 55 ◦C. The results indicated that Unc-LDH-CO3 and C-LDH-CO3 had an obvious protecting effect for acid phosphataseagainst excess energy which may destroy the functional group ofboth enzyme and substrate and then limit the enzymatic reactionrate.

3.4. Catalytic kinetics of free and immobilized acid phosphatase

The effects of substrate concentration on the reaction rate of freeand immobilized acid phosphatase were described well (R2 > 0.98,n = 6) by Michaelis–Menten equation: V = (Vmax S)/(Km + S), whereS is substrate concentration (mmol L−1), V is reaction rate andVmax is the maximum rate of reaction (�mol mg−1 min−1), Km isthe Michaelis constant (mmol L−1). The lower the Km value, thestronger the affinity of substrate for enzyme.

The Vmax value for free and immobilized acid phos-phatase on Unc-LDH-CO3 and C-LDH-CO3 was 0.68, 0.57 and0.62 �mol mg−1 min−1, respectively (Table 1). The Michaelisconstant (Km) for free enzyme was 1.09 mmol mL−1 while thatfor immobilized enzyme on Unc-LDH-CO3 and C-LDH-CO3 wasincreased to 1.22 and 1.19 mmol mL−1, respectively, suggestingthe decreased affinity of substrate for immobilized enzymes. Theincrease of Km value was also reported for acid phosphatase whenimmobilized on Ca-polygalacturonate and dry bead [4,31].

3.5. Stability of free and immobilized acid phosphatase

3.5.1. Thermal stabilityThe heat inactivation curves in Fig. 5 showed that free and

immobilized acid phosphatase on Unc-LDH-CO3 and C-LDH-CO3retained 32%, 56% and 46% of their initial activity after 150 min incu-bation at 50 ◦C. At 60 ◦C, the free and immobilized enzymes kepttheir initial activity to a level of 14%, 42% and 31%, respectively.Free acid phosphatase was nearly fully deactivated after 90 minincubation at 70 ◦C, while the immobilized enzyme on Unc-LDH-

CO3 and C-LDH-CO3 preserved 20% and 12% of their initial activityafter 150 min incubation at the same temperature.

The thermal deactivation of the free and immobilized enzymeswas fitted by the first-order kinetic equation to evaluate the deac-tivation rate: ln(A/A0) = −kdt + C1, where A0 and A are the initial

c-LDH-CO3 and I-E on C-LDH-CO3) at different pH (A) and temperature (B).

170 J. Zhu et al. / Colloids and Surfaces B: Biointerfaces 77 (2010) 166–173

e on LDHs (I-E on Unc-LDH-CO3 and I-E on C-LDH-CO3) at 50, 60, and 70 ◦C.

ar

pl(pTe

ptCvrffb4emiEowift

TT

Fig. 5. Thermal stability of free (F-E) and immobilized acid phosphatas

ctivity and the activity after t min incubation, kd is the deactivationate constant (min−1), C1 is a constant.

The deactivation energy of free and immobilized acid phos-hatase was then calculated according to Arrhenius equation:

n(kd) = −Ed/(RT) + C2, where kd is the deactivation rate constantmin−1), R is gas constant (8.314 J mol−1 K−1), T is absolute tem-erature, C1 is a constant, and Ed is deactivation energy (J mol−1).he lower the Ed value, the less the temperature sensitivity of thenzyme [13].

Good correlation coefficients (R2 > 0.95) were obtained and thearameters are listed in Table 2. The kd value was decreased inhe sequence of free enzyme > immobilized enzyme on C-LDH-O3 > immobilized enzyme on Unc-LDH-CO3. The half-life (t1/2)alue for free enzyme was 67, 28 and 9 min at 50 ◦C, 60 ◦C and 70 ◦C,espectively. The values were increased to 175, 114 and 44 minor immobilized enzyme on Unc-LDH-CO3 and 132, 81 and 33 minor immobilized enzyme on C-LDH-CO3. The Ed values for immo-ilized enzyme on Unc-LDH-CO3 and C-LDH-CO3 were 35.24 and0.66 kJ mol−1, which were significantly lower than that of freenzyme (65.44 kJ mol−1). These results suggested that the ther-al stability of acid phosphatase was enhanced markedly after the

mmobilization on LDH-CO3 particularly the uncalcined form. Thed value for acid phosphatase immobilized on composite beads

−1

f chitosan and activated clay was 66.4 kJ mol while that onet/dried pure chitosan beads, wet/dried chitosan–ZnO2 compos-

te beads ranged from 78 to 142 kJ mol−1 [13,14]. Lower Ed valuesor the immobilized acid phosphatase in the current study impliedhe less temperature sensitivity.

able 2hermal deactivation parameters of free (F-E) and immobilized acid phosphatase on LDH

Enzyme 50 ◦C 60 ◦C

Kd (min−1) t1/2 (min) Kd (min−1)

F-E 0.0059 67 0.0107I-E on Unc-LDH-CO3 0.0047 175 0.0063I-E on C-LDH-CO3 0.0039 132 0.0052

Fig. 6. Hydrolytic stability of free (F-E) and immobilized acid phosphatase on LDHs(I-E on Unc-LDH-CO3 and I-E on C-LDH-CO3).

3.5.2. Hydrolytic stabilityFree acid phosphatase retained 45%, 30% and 12% of its origi-

nal activity after the hydrolysis by proteinase for 1 h, 2 h and 24 h,respectively. However, 78%, 73% and 51% of the original activity waskept by immobilized enzyme on Unc-LDH-CO3 and 91%, 85% and64% was remained by immobilized enzyme on C-LDH-CO3 after thehydrolysis by proteinase for 1 h, 2 h and 24 h, respectively (Fig. 6).These data indicated clearly that acid phosphatase became more

resistant to proteolysis after the immobilization on LDH-CO3 espe-cially the calcined form. Similar increase in the hydrolytic stabilityof acid phosphatase on organo-mineral complex has been reportedby Rao et al. [32].

s (I-E on Unc-LDH-CO3 and I-E on C-LDH-CO3) at 50, 60, and 70 ◦C.

70 ◦C Ed (kJ mol−1)

t1/2 (min) Kd (min−1) t1/2 (min)

28 0.0245 9 65.44115 0.0114 44 35.24

81 0.0084 33 40.66

J. Zhu et al. / Colloids and Surfaces B: Biointerfaces 77 (2010) 166–173 171

sphat

bcciltt−tabthe

Fig. 7. ITC spectra for the hydrolysis of free (F-E) and immobilized acid pho

Hydrolytic heat of free and immobilized acid phosphatasey proteinase in 2 h was determined by ITC. The power–timeurves for hydrolytic samples (sample with acid phosphatase) andorresponding controls (sample without acid phosphatase) arellustrated in Fig. 7. The net hydrolytic enthalpy (�Hhyd) values areisted in Table 3. With the equal amount of enzyme (45 �g mL−1),he �Hhyd value for free acid phosphatase was −1204.3 �J, andhat for immobilized enzyme on Unc-LDH-CO3 and C-LDH-CO3 was528.3 and −453.9 �J, respectively. The negative values indicated

hat the hydrolysis of the enzyme was an exothermic process. Themount of hydrolytic heat is closely related to the hydrolytic sta-

ility of the enzyme. The higher the hydrolytic heat, the morehe enzyme molecules are hydrolyzed, and thus the lower theydrolytic stability [33]. The hydrolytic heat released from freenzyme was 2.3 and 2.7 times higher than that from immobilized

Table 3Heat released from the hydrolysis of free (F-E) and immobilizedacid phosphatase on LDHs (I-E on Unc-LDH-CO3 and I-E on C-LDH-CO3) (45 �g mL−1) by proteinase K in 2 h.

Enzyme Net �H (�J)

F-E −1204.3I-E on Unc-LDH-CO3 −528.3I-E on C-LDH-CO3 −453.9

ase on LDHs (I-E on Unc-LDH-CO3 and I-E on C-LDH-CO3) by proteinase K.

enzyme on Unc-LDH-CO3 and C-LDH-CO3, implying that hydrolyticstability of acid phosphatase was improved significantly after theimmobilization on LDH-CO3 particularly the calcined form. Thesefindings were in good agreement with the above chemical assays.

3.5.3. ReusabilityThe immobilized acid phosphatase on Unc-LDH-CO3 kept 62%

and 10% of the initial activity after 6 and 10 reuses, while 77% and30% of the initial activity was remained by the immobilized enzymeon C-LDH-CO3 after the same cycles (Fig. 8), indicating good dura-bility of acid phosphatase immobilized on LDH-CO3 especially thecalcined form. Higher reusability of acid phosphatase was reportedby the immobilization on 6-mercapto-chitin, which retained 80% ofits initial activity after 10 runs [18]. The immobilization of penicillinG acylase on calcined-Mg/Al-LDHs (Mg/Al = 2, calcined at 500 ◦C)also displayed higher activity (36%) even after 15 operational cycles[27]. However, acid phosphatases immobilized on LDH-CO3 in thecurrent study were much more durable than that on 2-mercapto-chitin, which showed a sharp reduction in the activity only after 3runs [18].

3.5.4. Storage stabilityAcid phosphatases immobilized on Unc-LDH-CO3 and C-LDH-

CO3 kept 40% and 48% of their initial activity after 90 days, whereasthe free enzyme lost all its activity after 60 days (Fig. 9). The semi-

172 J. Zhu et al. / Colloids and Surfaces B: B

Fig. 8. Reusability of immobilized acid phosphatase on LDHs (I-E on Unc-LDH-CO3

and I-E on C-LDH-CO3).

Fo

lgttpt[s

4

biiwwtoacb[tamiCa

5. Conclusions

ig. 9. Storage stability of free (F-E) and immobilized acid phosphatase on LDHs (I-En Unc-LDH-CO3 and I-E on C-LDH-CO3).

og plot of the relative activity against the duration of the daysave a half-life (t1/2) of 10 days for free enzyme, 55 and 77 days forhe enzyme immobilized on Unc-LDH-CO3 and C-LDH-CO3, respec-ively. These observations indicated that the storage stability of acidhosphatase was enhanced by the immobilization on LDH-CO3 par-icularly the calcined form. In an investigation by Rahman et al.15], lipase immobilized on Mg/Al-LDHs and their nanocompositeshowed storage stability up to 70% at 30 ◦C for 60 days.

. Discussions

The promotive effects for the thermal stability, hydrolytic sta-ility, reusability and storage stability of acid phosphatase by the

mmobilization on both Unc-LDH-CO3 and C-LDH-CO3 were shownn the present study. The enzyme immobilized on Unc-LDH-CO3

as more resistant to thermal inactivation than that on C-LDH-CO3,hile the enzyme immobilized on C-LDH-CO3 was more resistant

o proteolysis and more durable for reuse and storage than thatn Unc-LDH-CO3. The promotion in thermal stability of enzymefter the immobilization was usually ascribed to the loss of itsonformational flexibility [34], the immobilized enzyme thereforeecame rigid and remained stable even at higher temperature16]. Moreover, the stability of immobilized enzyme was propor-ional to the ratio of physical adsorption between the enzymend the support, which lock the enzyme into the active confor-

ation [16,35]. The iso-electric point (IEP) of acid phosphatase

s 5.0 [36], while the value of PZC for Unc-LDH-CO3 and C-LDH-O3 was 7.9 and 8.7, respectively. There is a strong electrostaticttraction between the negatively charged enzyme and positively

iointerfaces 77 (2010) 166–173

charged LDHs at the present study (pH 5.5). More rigid structureof immobilized acid phosphatase on both Unc-LDH-CO3 and C-LDH-CO3 compared to free enzyme may explain at least in partfor the increase of their thermal stability. However, the proportionof physical adsorption on the immobilization of acid phosphataseby C-LDH-CO3 might be more than that by Unc-LDH-CO3 becauseof the higher PZC and the increase of anion exchange capacity afterthe calcination of LDHs [24]. Higher thermal stability of acid phos-phatase immobilized on Unc-LDH-CO3 than that on C-LDH-CO3might be due to the decrease of heat capacity after the calcinationof LDHs. More heat energy from reaction or extra-environment wasabsorbed by Unc-LDH-CO3 and thus alleviated the destruction forthe immobilized enzyme. The results from XRD indicated that acidphosphatase was immobilized mainly on the external surface ofUnc-LDH-CO3 and C-LDH-CO3. According to SEM images, both sup-ports showed rough surface with three-dimensional pores (Fig. 2),acid phosphatase molecules could penetrate into these pores andbecome less accessible to proteinase [32,36]. Moreover, flocky mor-phology of C-LDH-CO3 was more porous than studded shape ofUnc-LDH-CO3. The steric hindrance for the diffusion of proteinaseon the surface of LDH-CO3 and the restriction for the penetrationof proteinase into the three-dimensional pores of the supports wasstronger on C-LDH-CO3 than that on Unc-LDH-CO3. More acid phos-phatase immobilized on C-LDH-CO3 than that on Unc-LDH-CO3thus was protected from hydrolysis by proteinase. The reusabil-ity of the immobilized enzyme was limited by the detachment ofenzyme from the supports and the distortion of active site of theenzyme after continuous use [37]. Larger amount of immobilizationand higher retention of residual activity might contribute togetherto the better durability for reuse of immobilized acid phosphataseon C-LDH-CO3 than that on Unc-LDH-CO3. The dissimilarity in ther-mal stability and the coincidence in hydrolytic stability suggestedthat better storage stability of immobilized acid phosphatase onC-LDH-CO3 than that on Unc-LDH-CO3 was ascribed chiefly to theadvantage in hydrolytic stability.

The affinity of substrate (pNPP) for the immobilized acid phos-phatase on LDH-CO3 was decreased compared to that for freeenzyme, which was in agreement with the loss of activity afterthe immobilization. Since the molecular size of pNPP is smallerthan proteinase [32], three-dimensional pores which prevented thepenetration of proteinase on the surface of LDH-CO3 may have littlelimitation for the diffusion of substrate to contact with immobilizedenzyme. Higher affinity of substrate for the enzyme immobilized onC-LDH-CO3 than that on Unc-LDH-CO3 might be due to the higherproportion of physical adsorption in the enzyme immobilization asdiscussed above, more enzyme molecules thus kept active confor-mation.

The optimal residual activity in the present study was 80% onUnc-LDH-CO3 and 88% on C-LDH-CO3. The values were higherthan that on most natural or synthesized supports such as redsoil colloids (39–72%), montmorillonite (20%), kaolinite (57%),goethite (68%), tannic acid (33%), composite beads of chitosan andactivated clay (41–61%), composite beads of chitosan and ZrO2powders (30–48%) which reported previously [12–14,32,36]. Actu-ally, there are abundant of Al–OH groups presented on the surfaceof LDH-CO3 [26], the benefit effects of these groups have beendocumented for the retention of activity of acid phosphatase andurease [32,38]. Consequently, LDH-CO3, particularly C-LDH-CO3, ispromising immobilization agent for acid phosphatase.

Layered double hydroxides of uncalcined- and calcined-Mg/Al-CO3 were used successfully as supports for the immobilization ofacid phosphatase by the means of direct adsorption. Little activ-

es B: B

iata

A

rF

R

[[[[

[[

[

[[

[[[[[[

[[[[[

[[[[33] M.L. Bianconi, Biophys. Chem. 126 (2007) 59–64.

J. Zhu et al. / Colloids and Surfac

ty loss, excellent thermal stability, hydrolytic stability, reusabilitynd storage stability of the immobilized acid phosphatases revealedheir promising potentials for practical application in the fields suchs agricultural production and environmental remediation.

cknowledgements

The research was financially supported by the National Natu-al Science Foundation of China (40825002) and the Internationaloundation for Science (C/2527-2).

eferences

[1] J.C. Tarafdar, N. Claassen, Biol. Fertil. Soils 5 (1988) 308.[2] J.C. Tarafdar, R.S. Yadav, S.C. Meena, J. Plant Nutr. Soil Sci. 164 (2001) 279.[3] G.A. Gilbert, J.D. Knight, Plant Cell Environ. 22 (1999) 801.[4] H.K. Pant, P.R. Warman, Biol. Fertil. Soils 30 (2000) 306.[5] L.J. Sikora, D.D. Kaufman, L.C. Horng, Biol. Fertil. Soils 9 (1990) 14.[6] F. Sannino, L. Gianfreda, Chemosphere 45 (2001) 417.[7] P. Bhattacharyya, S. Tripathy, K. Kim, S.H. Kim, Ecotoxicol. Environ. Saf. 71

(2008) 149.

[8] L. Gianfreda, M.A. Rao, Enzyme Microb. Technol. 35 (2004) 339.[9] C.F. Hoehamer, C.S. Mazur, N.L. Wolfe, J. Agric. Food. Chem. 53 (2005) 90.10] J.M. Sarkar, A. Leonowicz, J.M. Bollag, Soil Biol. Biochem. 21 (1989) 183.11] E. Kandeler, Biol. Fertil. Soils 9 (1990) 199.12] Q. Huang, W. Liang, P. Cai, Colloids Surf. B 45 (2005) 209.13] M.Y. Chang, R.S. Juang, Process Biochem. 39 (2004) 1087.

[[[[[

iointerfaces 77 (2010) 166–173 173

14] M.Y. Chang, R.S. Juang, Int. J. Biol. Macromol. 40 (2007) 224.15] M.B.A. Rahman, M. Basri, M.Z. Hussein, M.N.H. Idris, R.N.Z.R.A. Rahman, A.B.

Salleh, Catal. Today 93–95 (2004) 405.16] M.B.A. Rahman, U.H. Zaidan, M. Basri, M.Z. Hussein, R.N.Z.R.A. Rahman, A.B.

Salleh, J. Mol. Catal. B: Enzyme 50 (2008) 33.17] K. Ohmiya, S. Sugano, S.E. Yun, S. Shimizu, Agric. Biol. Chem. 47 (1983) 535.18] K. Kurita, H. Yoshino, S.I. Nishimura, S. Ishii, T. Mori, Y. Nishiyama, Carbohydr.

Polym. 32 (1997) 171.19] C.M. Lai, C.Y. Shin, J. Biomass Energy Soc. China 12 (1993) 31.20] F. Cavani, F. Trifiroand, A. Vaccari, Catal. Today 11 (1991) 173.21] S. Miyata, Clays Clay Miner. 23 (1975) 369.22] S. Miyata, A. Okada, Clays Clay Miner. 25 (1977) 14.23] S. Miyata, Clays Clay Miner. 28 (1980) 50.24] L. Châtelet, J.Y. Bottero, J. Yvon, A. Bouchelaghem, Colloids Surf. B 111 (1996)

167.25] C.D. Hoyo, Appl. Clay Sci. 36 (2007) 103.26] K.H. Goh, T.T. Lim, Z. Dong, Water Res. 42 (2008) 1343.27] L. Ren, J. He, D.G. Evans, X. Duan, R. Ma, J. Mol. Catal. B: Enzyme 16 (2001) 65.28] E. Geraud, V. Prevot, C. Forano, C. Mousty, Chem. Commun. 13 (2008) 1554.29] A. Violante, M. Pucci, V. Cozzolino, J. Zhu, M. Pigna, J. Colloid Interface Sci. 333

(2009) 63.30] Y. Xiong, Soil Colloids, vol. 2, Science Press, Beijing, 1985.31] C. Marzadori, C. Gessa, S. Ciurli, Biol. Fertil. Soils 27 (1998) 97.32] M.A. Rao, A. Violante, L. Gianfreda, Soil Biol. Biochem. 32 (2000) 1007.

34] G. Bayramoglu, E. Yalcin, M.Y. Arıca, Process Biochem. 40 (2005) 3505.35] J.P. Lenders, P. Germain, R.R. Crichton, Biotechnol. Bioeng. 27 (1985) 572.36] M.A. Rao, L. Gianfreda, F. Palmiero, A. Violante, Soil Sci. 161 (1996) 751.37] K.R.C. Reddy, A.M. Kayasha, J. Mol. Catal. B: Enzyme 38 (2006) 104.38] L. Gianfreda, M.A. Rao, A. Violante, Soil Sci. Soc. Am. J. 59 (1995) 805.