Colloids and Surfaces B: Biointerfaces 77 (2010) 166–173
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Colloids and Surfaces B: Biointerfaces
journal homepage: www.elsevier.com/locate/colsurfb
Immobilization of acid phosphatase on uncalcined and calcined Mg/Al-CO3
layered double hydroxides
Jun Zhu a,b , Qiaoyun Huang a,∗ , Massimo Pigna b , Antonio Violante b,∗∗
a
b
Key Laboratory of Subtropical Agriculture and Environment, Ministry of Agriculture, College of Resources and Environment, Huazhong Agricultural University, Wuhan 430070, China
Department of Soil, Plant, Environment and Animal Production Science, Faculty of Agriculture, University of Naples “Federico II”, Via University 100, 80055 Portici (Naples), Italy
a r t i c l e
i n f o
Article history:
Received 25 November 2009
Received in revised form
18 December 2009
Accepted 25 January 2010
Available online 4 February 2010
Keywords:
Acid phosphatase
Layered double hydroxides
Immobilization
Activity
Kinetics
Stability
a b s t r a c t
Acid phosphatase was immobilized on layered double hydroxides of uncalcined- and calcined-Mg/AlCO3 (Unc-LDH-CO3 , C-LDH-CO3 ) by the means of direct adsorption. Optimal pH and temperature for the
activity 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-LDHCO3 was increased to 1.22 and 1.19 mmol mL−1 , respectively, indicating the decreased affinity of substrate
for 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 lost
during 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 thermal
stability of acid phosphatase after the immobilization on LDH-CO3 especially the uncalcined form. Both
chemical assays and isothermal titration calorimetry (ITC) observations implied that hydrolytic stability
of acid phosphatase was promoted significantly after the immobilization on LDH-CO3 especially the
calcined form. Reusability investigation showed that more than 60% of the initial activity was remained
after six reuses of immobilized enzyme on Unc-LDH-CO3 and C-LDH-CO3 . A half-life (t1/2 ) of 10 days was
calculated 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 by
direct adsorption is an effective means and would have promising potential for the practical application
in agricultural production and environmental remediation.
© 2010 Elsevier B.V. All rights reserved.
1. Introduction
Acid phosphatase, which comes from microorganisms, animals, and plants, catalyzes the hydrolysis of a wide range of
phosphomonoesters and phosphoproteins nonspecifically in acidic
environments [1,2]. It plays a key role in the transformation of
organic phosphorus to orthophosphate, the only type of phosphorus source taken up by crops [3,4]. Acid phosphatase is also used as
an indicator enzyme for monitoring the risk of various pollutants
in soils [5–7]. Moreover, the enzyme is important in promoting
the degradation of organophosphorous pesticides in terrestrial and
aqueous environments [8,9].
It has been reported that free enzymes generally have a
short-lived activity because of rapid denaturation and degradation [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: [email protected] (Q. Huang), [email protected]
(A. Violante).
0927-7765/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.colsurfb.2010.01.020
to reuse or for improving their stability [13–16]. Ohmiya et al.
[17] have immobilized acid phosphatase on porous glass beads
for the dephosphorylation of casein. Kurita et al. [18] have compared the immobilization of acid phosphatase on 2-mercapto- and
6-mercapto-chitin, 6-mercapto-chitin was found to be a better support according to the durability of immobilized enzyme. Chang and
Juang [13] have taken composite beads of chitosan and activated
clay as support for the immobilization of acid phosphatase, the
immobilized enzyme kept 90% of its original activity after 50 times
of reuse. The immobilization of acid phosphatase on wet/dried
pure chitosan beads, wet/dried chitosan–ZnO2 composite beads
has also been observed by Chang and Juang [14], chitosan–ZnO2
composite as wet form was suggested as the most promising support for immobilization application. Moreover, acid phosphatase
has been immobilized on montmorillonite and chitosan by Lai and
Shin [19] to enhance the content of phosphorus in soils. However,
most of these reported carriers are organic or semi-organic matrices 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 acid
phosphatase by a type of inorganic clay, namely, layered double
hydroxides (LDHs).
J. Zhu et al. / Colloids and Surfaces B: Biointerfaces 77 (2010) 166–173
LDHs have a general formula of [M1−x 2+ Mx 3+ (OH)2 ]x+
[Ax/n n− ·mH2 O]x− , where M2+ , M3+ and An− are divalent cation,
trivalent cation and interlayer anion, respectively, x is defined
as M2+ /(M2+ + M3+ ) and varies between 0.20 and 0.33 [20,21].
These so-called anionic clays, which exist in nature or easily and
cheaply synthesized under laboratory conditions, consist of positively charged layers and negatively charged interlayer anions
[20,22]. The positive charges are developed due to the partial substitution of divalent metal ions by trivalent cations and balanced by
interlayer anions [20,23]. By calcination above 450 ◦ C, LDHs would
lose their layer structure due to dehydroxylation and decarbonation and form highly active composite metal oxides with larger
surface area, higher porosity and greater anion exchange capacity
[21,24]. Over the past decades, uncalcined- and calcined-LDHs have
been used in many fields such as pharmaceutics and environmental
remediation [25,26]. They have also been found to be available as
the host material for enzymes due to their charged structure and
porous surface. Ren et al. [27] reported that calcined-Mg/Al-CO3
have higher affinity for penicillin G acylase but lower percentage
of residual activity than calcined-Zn/Al-CO3 . Alkaline phosphatase
(AIP) immobilized within a Mg2 Al-LDH by “soft chemistry” coprecipitation synthesis showed 36–44% of residual activity [28]. The
thermal and storage stability of lipase was increased obviously
after the immobilization on layered double hydroxide of Zn/AlNO3 , Ni/Al-NO3 , Mg/Al-NO3 and Mg/Al-sodium dodecyl sulphate
(SDS) by direct adsorption [15,16], which is a physical method and
relatively easier and cheaper compare to chemical method [15].
The objectives of the present study are to investigate the
optimal conditions for the immobilization of acid phosphatase
by direct adsorption on Unc-LDH-CO3 and C-LDH-CO3 and the
kinetic property, thermal, hydrolytic, reusable and storage stability of the immobilized enzyme. The potentials of immobilized acid
phosphatase in the regulation of soil fertility and remediation of
pesticides pollution might be evaluated.
2. Materials and methods
2.1. Enzyme
Acid phosphatase (EC3.1.3.2 Type II, 1.0 units mg−1 from potato)
was purchased from Sigma Chemical Company.
2.2. Synthesis and characterization of uncalcined and calcined
LDH-CO3
The layered double hydroxide of Unc-LDH-CO3 was synthesized
by coprecipitation as described by Violante et al. [29]. Part of UncLDH-CO3 was calcined at 500 ◦ C in an oven for 4 h. Both Unc-LDHCO3 and C-LDH-CO3 were used for the following immobilization
studies.
The surface area of the sample was determined by high
speed automated surface area and pore size analyzer (Quantachrome Autosorb-1, USA), it was 90.5 m2 g−1 for Unc-LDH-CO3
and 176.8 m2 g−1 for C-LDH-CO3 . The point of zero charge (PZC)
was 7.9 for Unc-LDH-CO3 and 8.7 for C-LDH-CO3 as measured by
Mehlich method [30].
The SEM images of freeze dried LDHs–enzyme complexes were
obtained by a JSM-6390 scanning electron microscope (JEOL, Japan)
after the gold coating in vacuum by a JFC-1600 sputter coater
(JEOL, Japan). The X-ray diffraction (XRD) patterns of LDH-CO3 and
LDHs–enzyme complexes were obtained by a Rigaku diffractometer (Rigaku Corporation, Japan) equipped with Cu K␣ radiation.
2.3. Immobilization of acid phosphatase
In a 10 mL centrifuge tube, ten milligram of LDHs were mixed
with 4 mL of 10 mmol L−1 acetate buffer (pH 5.5) containing 200,
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 centrifuged at 10,000 × g for 20 min. The supernatant was collected
and the residue was washed twice by 1.5 mL of acetate buffer.
After centrifugation again, the washings were collected and the
enzyme on the final residue (LDHs–enzyme complex) was the
immobilized acid phosphatase. The concentration of acid phosphatase in the first supernatant and the following washings was
determined at 280 nm by UV-spectrophotometer using BSA as the
standard. The amount of acid phosphatase immobilized was calculated by subtracting the quantity of enzyme in solution from
enzyme added initially. The immobilization of acid phosphatase
was also investigated as a function of residence time and buffer
pH. The immobilized acid phosphatase which was prepared by
the immobilization of the enzyme with the initial concentration
of 250 ␮g mL−1 in 4 mL acetate buffer on 10 mg LDHs at pH 5.5 and
25 ◦ 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 were
determined as follows:
The pH-activity profiles were constructed by re-suspending the
LDHs–enzyme complex by 2 mL of 10 mmol L−1 buffer at various
pHs (acetate buffer for pH 4.5, 5.0, 5.5, 6.0, 6.5 and Tris–HCl buffer
for pH 7.0). One hundred microlitres of free acid phosphatase
(0.5 mg mL−1 ) or the thoroughly re-suspended LDHs–enzyme
complex was then mixed with 900 ␮L of corresponding buffer containing 8 ␮mol ␳-nitrophenyl phosphate (pNPP). After incubation
at 37 ◦ C for 1 h, the enzymatic reaction was terminated immediately by the addition of 1 mL of 1 mol L−1 NaOH solution [12]. The
concentration of enzymatic product ␳-nitrophenol was determined
directly at 405 nm spectrophotometrically. The specific activity of
free and immobilized acid phosphatase was defined as ␮mol ␳nitrophenol released by the catalysis of 1 mg acid phosphatase
within 1 min.
The temperature–activity profiles were constructed by conducting 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 enzyme
in 10 mmol L−1 of acetate buffer (pH 5.5) containing 1, 2, 4, 6, and
8 ␮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 ␮L
of free enzyme (0.5 mg mL−1 ) or the thoroughly re-suspended
LDHs–enzyme complex in the absence of substrate at 50, 60, and
70 ◦ C for 30, 60, 90, 120, and 150 min. The activity was then determined after the further incubation with 900 ␮L of 10 mmol L−1 of
acetate buffer (pH 5.5) containing 8 ␮mol pNPP at 37 ◦ C for 1 h.
The hydrolytic stability was estimated by mixing 100 ␮L
of free enzyme (0.5 mg mL−1 ) or the thoroughly re-suspended
LDHs–enzyme complex with 900 ␮L of 10 mmol L−1 of acetate
buffer (pH 5.5) containing 25 ␮g of proteinase K. The mixture was
thoroughly dispersed and then incubated at 37 ◦ C for 1 h, 2 h and
24 h, respectively. One millilitre of 10 mmol L−1 of acetate buffer
(pH 5.5) containing 8 ␮mol pNPP was then added immediately
into the tube and the mixture was incubated in succession at
37 ◦ C for 1 h. No proteinase exposure was tested as the control
for free and immobilized enzyme. The hydrolytic stability of free
and immobilized phosphatase was calculated according to the following expression: hydrolytic stability (%) = (specific activity of acid
phosphatase in the presence of proteinase/specific activity of acid
phosphatase in the absence of proteinase) × 100. The hydrolytic
stability of free and immobilized enzyme was also investigated by
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J. Zhu et al. / Colloids and Surfaces B: Biointerfaces 77 (2010) 166–173
Fig. 1. Immobilization of acid phosphatase on LDHs with increasing enzyme concentrations (A), reaction time (B) and buffer pH (C).
ITC with an isothermal microcalorimeter TAM III (Thermometric
AB, Sweden). Net hydrolytic heat was calculated by deducting the
dilution and/or adsorption heat of proteinase (sample without acid
phosphatase) from the total heat (sample with acid phosphatase).
For reusability, both immobilized enzyme were employed to
catalyze the hydrolysis of pNPP repeatedly in a continuous cycles.
The storage stability was observed by storing the free and immobilized enzymes in 10 mmol L−1 of acetate buffer (pH 5.5) at 4 ◦ C for
5, 10, 30, 60, and 90 days. The residual activity was determined in
time by the method described above.
of the enzyme on both supports reached a plateau value at an
enzyme concentration of 250 ␮g mL−1 within 2 h. The maximum
immobilization was observed at pH 5.5. The percentage of acid
phosphatase immobilized at optimal conditions by Unc-LDH-CO3
and C-LDH-CO3 was 59% and 73%, respectively, indicating higher
immobilization of acid phosphatase on the latter support than that
on the former one.
3. Results
The LDHs and/or LDHs–enzyme complexes were characterized
by SEM and XRD techniques. A flocky morphology of C-LDH-CO3
and a studded shape of Unc-LDH-CO3 after the immobilization of
acid phosphatase were recorded by SEM (Fig. 2). The XRD patterns
of Unc-LDH-CO3 in Fig. 3 showed some typical peaks which characterized the widely reported hydrotalcite [29], a basal space of
0.756 nm was calculated from the 003 reflection. For C-LDH-CO3 ,
3.1. Immobilization of acid phosphatase on LDHs
Figure 1 depicts the amount of acid phosphatase immobilized by Unc-LDH-CO3 and C-LDH-CO3 with increasing enzyme
concentrations, reaction time and buffer pH. The immobilization
3.2. Characterization of LDHs and LDHs–acid phosphatase
complex
Fig. 2. SEM micrographs of Unc-LDH-CO3 (A) and C-LDH-CO3 (B) after enzyme immobilization.
J. Zhu et al. / Colloids and Surfaces B: Biointerfaces 77 (2010) 166–173
169
Table 1
Kinetic parameters of free (F-E) and immobilized acid phosphatase on LDHs (I-E on
Unc-LDH-CO3 and I-E on C-LDH-CO3 ) by Michaelis–Menten equation.
Enzyme
Km (mmol mL−1 )
Vmax (␮mol mg−1 min−1 )
F-E
I-E on Unc-LDH-CO3
I-E on C-LDH-CO3
1.09
1.22
1.19
0.68
0.57
0.62
acid phosphatase immobilized on LDH-CO3 exceeded that of free
enzyme at 55 ◦ C. The results indicated that Unc-LDH-CO3 and CLDH-CO3 had an obvious protecting effect for acid phosphatase
against excess energy which may destroy the functional group of
both enzyme and substrate and then limit the enzymatic reaction
rate.
Fig. 3. Powder XRD patterns of Unc-LDH-CO3 and C-LDH-CO3 before and after
enzyme immobilization.
the typical peaks of hydrotalcite disappeared while new broad
peaks appeared (Fig. 3), indicating the collapse of layered double structure and the formation of Mg–Al mixed oxides. The XRD
patterns of both Unc-LDH-CO3 –enzyme and C-LDH-CO3 –enzyme
complex were similar to that of the corresponding pure mineral
(Fig. 3), suggesting that acid phosphatase was limited to be intercalated into Unc-LDH-CO3 and the immobilization of the enzyme
on the surface of C-LDH-CO3 prevented its structural reconstruction. Therefore, acid phosphatase was immobilized mainly on the
external surface of Unc-LDH-CO3 and C-LDH-CO3 .
3.3. Activity of free and immobilized acid phosphatase
As revealed by the pH-activity profiles (Fig. 4A), the maximum
activity for free and immobilized acid phosphatase were observed
at pH 5.5, where the enzyme on Unc-LDH-CO3 and C-LDH-CO3
retained 80 and 88% of the residual activity. These results indicated
that the optimal pH for the activity of acid phosphatase did not
change after the immobilization. The optimal pH for the activity
of acid phosphatase was also reported at pH 5.5 and kept constant
after the immobilization on Red soil colloids, kaolinite, goethite and
6-mercapto-chitin [12,18]. However, a shift of optimal pH from 5.2
for free acid phosphatase to 2.9 for the immobilized enzyme on
composite beads of chitosan and ZrO2 powders was documented
by Chang and Juang [13].
Figure 4B presents the activity of free and immobilized acid
phosphatase at temperatures from 15 ◦ C to 60 ◦ C. The activity of
free and immobilized enzyme increased with the increase of temperature from 15 ◦ C to 37 ◦ C and reached maximum at 37 ◦ C. Free
enzyme lost its activity rapidly above 37 ◦ C, whereas enzymes
immobilized on both supports were more stable. The activity of
3.4. Catalytic kinetics of free and immobilized acid phosphatase
The effects of substrate concentration on the reaction rate of free
and immobilized acid phosphatase were described well (R2 > 0.98,
n = 6) by Michaelis–Menten equation: V = (Vmax S)/(Km + S), where
S is substrate concentration (mmol L−1 ), V is reaction rate and
Vmax is the maximum rate of reaction (␮mol mg−1 min−1 ), Km is
the Michaelis constant (mmol L−1 ). The lower the Km value, the
stronger the affinity of substrate for enzyme.
The Vmax value for free and immobilized acid phosphatase on Unc-LDH-CO3 and C-LDH-CO3 was 0.68, 0.57 and
0.62 ␮mol mg−1 min−1 , respectively (Table 1). 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, suggesting
the decreased affinity of substrate for immobilized enzymes. The
increase of Km value was also reported for acid phosphatase when
immobilized on Ca-polygalacturonate and dry bead [4,31].
3.5. Stability of free and immobilized acid phosphatase
3.5.1. Thermal stability
The heat inactivation curves in Fig. 5 showed that free and
immobilized acid phosphatase on Unc-LDH-CO3 and C-LDH-CO3
retained 32%, 56% and 46% of their initial activity after 150 min incubation at 50 ◦ C. At 60 ◦ C, the free and immobilized enzymes kept
their initial activity to a level of 14%, 42% and 31%, respectively.
Free acid phosphatase was nearly fully deactivated after 90 min
incubation at 70 ◦ C, while the immobilized enzyme on Unc-LDHCO3 and C-LDH-CO3 preserved 20% and 12% of their initial activity
after 150 min incubation at the same temperature.
The thermal deactivation of the free and immobilized enzymes
was fitted by the first-order kinetic equation to evaluate the deactivation rate: ln(A/A0 ) = −kd t + C1 , where A0 and A are the initial
Fig. 4. Activity of free (F-E) and immobilized acid phosphatase on LDHs (I-E on Unc-LDH-CO3 and I-E on C-LDH-CO3 ) at different pH (A) and temperature (B).
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J. Zhu et al. / Colloids and Surfaces B: Biointerfaces 77 (2010) 166–173
Fig. 5. Thermal stability of free (F-E) and immobilized acid phosphatase on LDHs (I-E on Unc-LDH-CO3 and I-E on C-LDH-CO3 ) at 50, 60, and 70 ◦ C.
activity and the activity after t min incubation, kd is the deactivation
rate constant (min−1 ), C1 is a constant.
The deactivation energy of free and immobilized acid phosphatase was then calculated according to Arrhenius equation:
ln(kd ) = −Ed /(RT) + C2 , where kd is the deactivation rate constant
(min−1 ), R is gas constant (8.314 J mol−1 K−1 ), T is absolute temperature, C1 is a constant, and Ed is deactivation energy (J mol−1 ).
The lower the Ed value, the less the temperature sensitivity of the
enzyme [13].
Good correlation coefficients (R2 > 0.95) were obtained and the
parameters are listed in Table 2. The kd value was decreased in
the sequence of free enzyme > immobilized enzyme on C-LDHCO3 > immobilized enzyme on Unc-LDH-CO3 . The half-life (t1/2 )
value for free enzyme was 67, 28 and 9 min at 50 ◦ C, 60 ◦ C and 70 ◦ C,
respectively. The values were increased to 175, 114 and 44 min
for immobilized enzyme on Unc-LDH-CO3 and 132, 81 and 33 min
for immobilized enzyme on C-LDH-CO3 . The Ed values for immobilized enzyme on Unc-LDH-CO3 and C-LDH-CO3 were 35.24 and
40.66 kJ mol−1 , which were significantly lower than that of free
enzyme (65.44 kJ mol−1 ). These results suggested that the thermal stability of acid phosphatase was enhanced markedly after the
immobilization on LDH-CO3 particularly the uncalcined form. The
Ed value for acid phosphatase immobilized on composite beads
of chitosan and activated clay was 66.4 kJ mol−1 while that on
wet/dried pure chitosan beads, wet/dried chitosan–ZnO2 composite beads ranged from 78 to 142 kJ mol−1 [13,14]. Lower Ed values
for the immobilized acid phosphatase in the current study implied
the less temperature sensitivity.
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 stability
Free acid phosphatase retained 45%, 30% and 12% of its original activity after the hydrolysis by proteinase for 1 h, 2 h and 24 h,
respectively. However, 78%, 73% and 51% of the original activity was
kept by immobilized enzyme on Unc-LDH-CO3 and 91%, 85% and
64% was remained by immobilized enzyme on C-LDH-CO3 after the
hydrolysis 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 especially the calcined form. Similar increase in the hydrolytic stability
of acid phosphatase on organo-mineral complex has been reported
by Rao et al. [32].
Table 2
Thermal deactivation parameters of free (F-E) and immobilized acid phosphatase on LDHs (I-E on Unc-LDH-CO3 and I-E on C-LDH-CO3 ) at 50, 60, and 70 ◦ C.
Enzyme
50 ◦ C
60 ◦ C
−1
Kd (min
F-E
I-E on Unc-LDH-CO3
I-E on C-LDH-CO3
0.0059
0.0047
0.0039
)
70 ◦ C
−1
t1/2 (min)
Kd (min
67
175
132
0.0107
0.0063
0.0052
)
Ed (kJ mol−1 )
−1
t1/2 (min)
Kd (min
28
115
81
0.0245
0.0114
0.0084
)
t1/2 (min)
9
44
33
65.44
35.24
40.66
J. Zhu et al. / Colloids and Surfaces B: Biointerfaces 77 (2010) 166–173
171
Fig. 7. ITC spectra for the hydrolysis of free (F-E) and immobilized acid phosphatase on LDHs (I-E on Unc-LDH-CO3 and I-E on C-LDH-CO3 ) by proteinase K.
Hydrolytic heat of free and immobilized acid phosphatase
by proteinase in 2 h was determined by ITC. The power–time
curves for hydrolytic samples (sample with acid phosphatase) and
corresponding controls (sample without acid phosphatase) are
illustrated in Fig. 7. The net hydrolytic enthalpy (Hhyd ) values are
listed in Table 3. With the equal amount of enzyme (45 ␮g mL−1 ),
the Hhyd value for free acid phosphatase was −1204.3 ␮J, and
that for immobilized enzyme on Unc-LDH-CO3 and C-LDH-CO3 was
−528.3 and −453.9 ␮J, respectively. The negative values indicated
that the hydrolysis of the enzyme was an exothermic process. The
amount of hydrolytic heat is closely related to the hydrolytic stability of the enzyme. The higher the hydrolytic heat, the more
the enzyme molecules are hydrolyzed, and thus the lower the
hydrolytic stability [33]. The hydrolytic heat released from free
enzyme was 2.3 and 2.7 times higher than that from immobilized
Table 3
Heat released from the hydrolysis of free (F-E) and immobilized
acid phosphatase on LDHs (I-E on Unc-LDH-CO3 and I-E on CLDH-CO3 ) (45 ␮g mL−1 ) by proteinase K in 2 h.
Enzyme
F-E
I-E on Unc-LDH-CO3
I-E on C-LDH-CO3
Net H (␮J)
−1204.3
−528.3
−453.9
enzyme on Unc-LDH-CO3 and C-LDH-CO3 , implying that hydrolytic
stability of acid phosphatase was improved significantly after the
immobilization on LDH-CO3 particularly the calcined form. These
findings were in good agreement with the above chemical assays.
3.5.3. Reusability
The immobilized acid phosphatase on Unc-LDH-CO3 kept 62%
and 10% of the initial activity after 6 and 10 reuses, while 77% and
30% of the initial activity was remained by the immobilized enzyme
on C-LDH-CO3 after the same cycles (Fig. 8), indicating good durability of acid phosphatase immobilized on LDH-CO3 especially the
calcined form. Higher reusability of acid phosphatase was reported
by the immobilization on 6-mercapto-chitin, which retained 80% of
its initial activity after 10 runs [18]. The immobilization of penicillin
G 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 the
current study were much more durable than that on 2-mercaptochitin, which showed a sharp reduction in the activity only after 3
runs [18].
3.5.4. Storage stability
Acid phosphatases immobilized on Unc-LDH-CO3 and C-LDHCO3 kept 40% and 48% of their initial activity after 90 days, whereas
the free enzyme lost all its activity after 60 days (Fig. 9). The semi-
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J. Zhu et al. / Colloids and Surfaces B: Biointerfaces 77 (2010) 166–173
Fig. 8. Reusability of immobilized acid phosphatase on LDHs (I-E on Unc-LDH-CO3
and I-E on C-LDH-CO3 ).
Fig. 9. Storage stability of free (F-E) and immobilized acid phosphatase on LDHs (I-E
on Unc-LDH-CO3 and I-E on C-LDH-CO3 ).
log plot of the relative activity against the duration of the days
gave a half-life (t1/2 ) of 10 days for free enzyme, 55 and 77 days for
the enzyme immobilized on Unc-LDH-CO3 and C-LDH-CO3 , respectively. These observations indicated that the storage stability of acid
phosphatase was enhanced by the immobilization on LDH-CO3 particularly the calcined form. In an investigation by Rahman et al.
[15], lipase immobilized on Mg/Al-LDHs and their nanocomposites
showed storage stability up to 70% at 30 ◦ C for 60 days.
4. Discussions
The promotive effects for the thermal stability, hydrolytic stability, reusability and storage stability of acid phosphatase by the
immobilization on both Unc-LDH-CO3 and C-LDH-CO3 were shown
in the present study. The enzyme immobilized on Unc-LDH-CO3
was more resistant to thermal inactivation than that on C-LDH-CO3 ,
while the enzyme immobilized on C-LDH-CO3 was more resistant
to proteolysis and more durable for reuse and storage than that
on Unc-LDH-CO3 . The promotion in thermal stability of enzyme
after the immobilization was usually ascribed to the loss of its
conformational flexibility [34], the immobilized enzyme therefore
became rigid and remained stable even at higher temperature
[16]. Moreover, the stability of immobilized enzyme was proportional to the ratio of physical adsorption between the enzyme
and the support, which lock the enzyme into the active conformation [16,35]. The iso-electric point (IEP) of acid phosphatase
is 5.0 [36], while the value of PZC for Unc-LDH-CO3 and C-LDHCO3 was 7.9 and 8.7, respectively. There is a strong electrostatic
attraction between the negatively charged enzyme and positively
charged LDHs at the present study (pH 5.5). More rigid structure
of immobilized acid phosphatase on both Unc-LDH-CO3 and CLDH-CO3 compared to free enzyme may explain at least in part
for the increase of their thermal stability. However, the proportion
of physical adsorption on the immobilization of acid phosphatase
by C-LDH-CO3 might be more than that by Unc-LDH-CO3 because
of the higher PZC and the increase of anion exchange capacity after
the calcination of LDHs [24]. Higher thermal stability of acid phosphatase immobilized on Unc-LDH-CO3 than that on C-LDH-CO3
might be due to the decrease of heat capacity after the calcination
of LDHs. More heat energy from reaction or extra-environment was
absorbed by Unc-LDH-CO3 and thus alleviated the destruction for
the immobilized enzyme. The results from XRD indicated that acid
phosphatase was immobilized mainly on the external surface of
Unc-LDH-CO3 and C-LDH-CO3 . According to SEM images, both supports showed rough surface with three-dimensional pores (Fig. 2),
acid phosphatase molecules could penetrate into these pores and
become less accessible to proteinase [32,36]. Moreover, flocky morphology of C-LDH-CO3 was more porous than studded shape of
Unc-LDH-CO3 . The steric hindrance for the diffusion of proteinase
on the surface of LDH-CO3 and the restriction for the penetration
of proteinase into the three-dimensional pores of the supports was
stronger on C-LDH-CO3 than that on Unc-LDH-CO3 . More acid phosphatase immobilized on C-LDH-CO3 than that on Unc-LDH-CO3
thus was protected from hydrolysis by proteinase. The reusability of the immobilized enzyme was limited by the detachment of
enzyme from the supports and the distortion of active site of the
enzyme after continuous use [37]. Larger amount of immobilization
and higher retention of residual activity might contribute together
to the better durability for reuse of immobilized acid phosphatase
on C-LDH-CO3 than that on Unc-LDH-CO3 . The dissimilarity in thermal stability and the coincidence in hydrolytic stability suggested
that better storage stability of immobilized acid phosphatase on
C-LDH-CO3 than that on Unc-LDH-CO3 was ascribed chiefly to the
advantage in hydrolytic stability.
The affinity of substrate (pNPP) for the immobilized acid phosphatase on LDH-CO3 was decreased compared to that for free
enzyme, which was in agreement with the loss of activity after
the immobilization. Since the molecular size of pNPP is smaller
than proteinase [32], three-dimensional pores which prevented the
penetration of proteinase on the surface of LDH-CO3 may have little
limitation for the diffusion of substrate to contact with immobilized
enzyme. Higher affinity of substrate for the enzyme immobilized on
C-LDH-CO3 than that on Unc-LDH-CO3 might be due to the higher
proportion of physical adsorption in the enzyme immobilization as
discussed above, more enzyme molecules thus kept active conformation.
The optimal residual activity in the present study was 80% on
Unc-LDH-CO3 and 88% on C-LDH-CO3 . The values were higher
than that on most natural or synthesized supports such as red
soil colloids (39–72%), montmorillonite (20%), kaolinite (57%),
goethite (68%), tannic acid (33%), composite beads of chitosan and
activated clay (41–61%), composite beads of chitosan and ZrO2
powders (30–48%) which reported previously [12–14,32,36]. Actually, there are abundant of Al–OH groups presented on the surface
of LDH-CO3 [26], the benefit effects of these groups have been
documented for the retention of activity of acid phosphatase and
urease [32,38]. Consequently, LDH-CO3 , particularly C-LDH-CO3 , is
promising immobilization agent for acid phosphatase.
5. Conclusions
Layered double hydroxides of uncalcined- and calcined-Mg/AlCO3 were used successfully as supports for the immobilization of
acid phosphatase by the means of direct adsorption. Little activ-
J. Zhu et al. / Colloids and Surfaces B: Biointerfaces 77 (2010) 166–173
ity loss, excellent thermal stability, hydrolytic stability, reusability
and storage stability of the immobilized acid phosphatases revealed
their promising potentials for practical application in the fields such
as agricultural production and environmental remediation.
Acknowledgements
The research was financially supported by the National Natural Science Foundation of China (40825002) and the International
Foundation for Science (C/2527-2).
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