Huang - adsorção e dessorção de fosfatase

7/27/2019 Huang - adsoro e dessoro de fosfatase

1/6

Colloids and Surfaces B: Biointerfaces 45 (2005) 209214

Adsorption, desorption and activities of acid phosphataseon various colloidal particles from an Ultisol

Qiaoyun Huang, Wei Liang, Peng Cai

State Key Laboratory of Agricultural Microbiology, Faculty of Resources and Environment,

Huazhong Agriculture University, Wuhan 430070, China

Received 8 July 2005; received in revised form 28 July 2005; accepted 22 August 2005

Abstract

Adsorption, desorption and activity of acid phosphatase on various soil colloidal particles andpure clay minerals were studied. Higher adsorption

amounts and low percentage of desorption of acid phosphatase were found on fine soil clays (


2/6

210 Q. Huang et al. / Colloids and Surfaces B: Biointerfaces 45 (2005) 209214

with pure kaolinite and synthetic iron oxide. The role of natu-

ral humic substance in enzyme adsorption and its influence on

enzyme activity and stability is to be clarified. We intended to

provide insight into the interactive mechanisms between natural

soil particles and enzymes. Information in this respect is essen-

tial in understanding the behavior and activities of enzymes in

soil and sediments.

2. Materials and methods

2.1. Enzyme

Acid phosphatase (EC 3.1.3.2, type II, 0.8 units mg1, from

potato) was purchased from Sigma.

2.2. Preparation of soil colloids and minerals

A Red soil (Ultisol, USDA classification) was sampled from

the 11 to 40 cm layer of an upland in Wenquan, Hubei province,

China. After removal of coarse organic residue, the soil wasrinsed in deionized water anddispersed by adding 0.01 M NaOH

solution dropwise to pH 78 together with sonication. Two soil

colloidal components, i.e. fine clay (


3/6

Q. Huang et al. / Colloids and Surfaces B: Biointerfaces 45 (2005) 209214 211

and calculated as described above. Sequential desorption of acid

phosphatase from soil colloid and mineralenzyme complexes

was then carried out using 3 mL of 2 mol L1 NaCl (pH 5.5) and

3mL of 0.1molL1 phosphate solution (pH 5.5). Desorption

lasted for 1 h at 25 C for each agent. The supernatant was col-

lected and the concentration of enzyme was measured at 280 nm

by spectrophotometry. The percent desorption from soil clays

and minerals for each agent was calculated according to the

amount of enzyme bound and desorbed.

2.5. Enzyme assay

In 10 mL centrifuge tube, 0.5 mL of 10 mg L1 soil colloid

suspension was mixed with 1 mL 0.3 mol L1 acetate buffer (pH

5.5), 1.2 mL deionized water and 0.3 mL 2 mg mL1 acid phos-

phates solution. The mixture was shaken at 25 C for 2 h and

centrifuged at 30,000 g for 15 min. The supernatant was col-

lected. The residue was washed twice with 1.5mL acetate buffer

and the washings were combined with the first supernatant. The

concentration of enzyme in the solution was determined directlyby spectrophotometry at 280 nm. The amount of enzyme bound

on various soil colloids was calculated. The residue was resus-

pended in 3 mL of acetate buffer. For enzyme assay, 0.03 mL

of thoroughly mixed enzymecolloid mixture was mixed with

1 mL acetate buffer containing 6 mM -nitrophenyl phosphate

and incubated at 37 C for 1 h. The enzyme reaction was termi-

nated by the addition of 1mL of 1 mol L1 NaOH solution. The

concentration of enzymatic product -nitrophenol was analyzed

spectrophotometrically at 405 nm. The specific activities of free

and immobilized enzymes were expressed as g -nitrophenol

catalyzed by 1 mg of enzyme within 1 h. The activities of free

and immobilized enzymes were also measured at pH 4.0, 4.5,5.0, 5.5, 6.0, 6.5 and 7.0. The pH was controlled by acetate

(4.06.0) and phosphate buffers (6.07.0). The thermal stability

of free and immobilized enzymes was examined by analyzing

their activities at elevated temperatures from 15 to 85 C.

All the experiments were conducted in triplicate.

3. Results and discussions

3.1. Adsorption on soil colloid, kaolinite and goethite

Fig. 1 shows that the adsorption curves of acid phosphatase

on soil colloids, kaolinite and goethite are typical L curves [22].

The amount of adsorption increased gradually with the increase

of enzyme concentration in the solution.

The adsorption data fitted well Langmuir equation

(R2 > 0.96):

y =BmaxKx

1 +Kx.

The calculated parameters adsorption capacity (Bmax) and

binding affinity (K) are listed in Table 2. The results revealed

that goethite adsorbed the largest amounts of enzyme among

the soil colloids and minerals examined. Kaolinite had the

least adsorption capacity. Fine soil clays adsorbed significantly

greater amount of enzyme than coarse soil clays. For the same

Fig. 1. Adsorption isotherms of acid phosphatase on soil colloids and clay

minerals.

particle size of clay fractions, the calculated adsorption capacity

for organic clays (623.6 mg/g) was larger than that for inorganic

clays (594.4 mg/g). The facilitated effects of organic substances

in adsorption of enzymes have been conformed by quite a num-

ber of studies [18]. Soil humic compounds may adsorb sub-

stantial enzymes by ion exchange, covalent complexation and

hydrogen bonding [4]. It was assumed that the enzymes were

trapped within the macromolecular net of the humic acids and

also immobilized at the surface by adsorption forces [1].

The different capacities of soil clays to adsorb acid phos-

phatase are due to their discrepancies in mineral and organic

components as well as surface properties. The higher amount of

Table 2

Langmuir parameters for adsorption of acid phosphatase on soil colloids and

clay minerals

Colloid type Bmax (g mg1) K R

Fine inorganic clay 594.4 17.7 0.993

Fine organic clay 623.6 11.3 0.994

Coarse inorganic clay 248.6 58.6 0.992

Coarse organic clay 305.3 37.8 0.997

Kaolinite 154.5 46.8 0.978

Goethite 848.0 10.8 0.996

K, constant related to the binding energy; Bmax, maximum adsorption capacity;

R, correlation coefficient.


4/6


enzymes adsorbed by fine soil clays is attributed to the higher

content of iron oxides, and larger surface area. Fine clays also

have a large cation exchange capacity. This would suggest that

ion exchange process plays an important role in the adsorption

of enzyme on soil clays studied. Ligand exchange may account

for the large adsorption by goethite. The isoelectric point (iep)

for goethite was 8.27 [23]. The iep for acid phosphatase was 5.0

[12]. The enzyme can also be adsorbed on goethite via electro-

static interactions because goethite was positively charged and

phosphatase was negatively charged in the present experiment

(pH 5.5). Our result would suggest that acid phosphates tend

to bind on finer colloidal particles in acidic soil environments.

Organic components would enhance this adsorption process.

The Kvalue is related to the binding energy between enzyme

molecules and the solid surface. The greater the K value, the

higher the affinity. Our data show that kaolinite and coarse soil

clays had a higher affinity for enzyme molecules than fine soil

clays and goethite. The lower affinity of fine clays for enzyme is

presumably attributed to their higher contents of iron oxides. It

appears that thebinding affinity of soil clay to enzymemoleculeswas inhibited by the presence of organic matter.

3.2. Desorption of bound enzymes

Fig. 2 shows that the percent desorptions of enzyme from the

systems of goethite, fine clays, coarse clays by 2 mol L1 NaCl

were 15.4, 2330.7 and 47.162.2%, respectively. Only 2.1% of

adsorbed enzyme was leased by NaCl from kaolinite. Phosphate

desorbed 31.7% of enzyme from goethite, 13.517.8% from

soil clays and 5% from kaolinite. Proteins can be adsorbed on

soil particles via ion exchange process [19]. The molecules

desorbed by NaCl were usually regarded as exchangeable, whilethose removed by phosphate were considered as specifically

adsorbed (ligand exchange form) [23,24]. From these results, it

is obvious that large amount of enzyme molecules on goethite

were adsorbed via ligand exchange process. More than 50% of

enzymes were adsorbed on coarse soil clays electrostatically.

Fig. 2. Desorption of acid phosphatase from various complexes by NaCl and

phosphate.

On fine clays exchangeable enzymes amounted to 2331%.

The percentage of specifically adsorbed enzyme on various soil

clays was from 13 to 18%. For kaolinite, the proportions for

both exchangeable and specifically adsorbed enzymes are less

than 5%.

It is certain that, besides ion and ligand exchange, there are

some other interactions for the binding of enzymes with clay

minerals, such as van der Waals force, hydrophobic force and

hydrogen bonding [25]. In the present study, the function of the

exchangeablecation for enzyme molecules andvarious soilclays

seemed to be paramount in the different interactions, includ-

ing direct coordination of polar groups (carboxyl, carbonyl or

amino) to the exchangeable cation or indirect coordination to the

exchangeable cation through a water bridge. In addition, the

protonatedaminogroup(NH3+) is an excellenthydrogen-bond

donor, and it can form a hydrogen-bond with the structural oxy-

gen of siloxane surfaces [26]. Some of these bindings may not

be destroyed by NaCl or phosphate. Therefore, a large propor-

tion of enzyme molecules were still adsorbed on soil clays after

the washing of phosphate. Especially for kaolinite the major-ity of enzymes could not be removed by NaCl and phosphate,

indicating van der Waals force and hydrogen bonding may play

important roles in the adsorption of enzyme. Although soil clays

studied also contain large amounts of kaolinite, the mineral may

be present in oxide-coated form or complexed by organic com-

ponents.

The data revealed that for soil clays with the same particle

size, more enzyme molecules were adsorbed on organic frac-

tions, suggesting more enzymes are adsorbed on organic soil

components via electrostatic force. No remarkable differences

were observed for the amount of specifically adsorbed enzymes

on inorganic and organic clays. Enzymes released by NaCl areadsorbed weakly on the solid surface. Therefore, it is evident

that in comparison to fine clays more loosely bound enzymes

were attached on coarse soil clays. Enzymes on soil organic

components are not tightly bound and can be easily removed.

3.3. Residual activity of enzyme complexes

Table 3 shows that the specific activity of free acid phos-

phatase is 2556gPNPmg1 h1. Enzyme on thefine inorganic

and organic soil clay remained 72 and 61% of activity, respec-

tively. The residual activity for the coarse soil clay is 3944%.

Enzyme bound on goethite and kaolinite retained 68 and 57%

of residual activity. These results suggested that enzymes on

Table 3

Activities of soil colloidsacid phosphatase complexes

Systems Specific activity (g

-nitrophenol mg1 h1)

Residual activity (%)

Fine inorganic clay 1846 72.2

Fine organic clay 1551 60.7

Coarse inorganic clay 1126 44.0

Coarse organic clay 998 39.1

Goethite 1731 67.7

Kaolinite 1467 57.4

Free enzyme 2556


5/6

Q. Huang et al. / Colloids and Surfaces B: Biointerfaces 45 (2005) 209214 213

fine soil particle retained higher activities. Enzyme activity was

inhibited in the presence of organic matter. The inhibition of

phosphatase activity by synthetic and natural humic compounds

was reported previously by several investigators [6,27]. Several

mechanisms have been proposed for the inhibitory action of

humic acids on enzyme activity, such as (1) complexation by

humic acids or the metal ions that are part of the structure of

the active sites; (2) conformational change in the enzymes; (3)

competition with substrate for the catalytically active site and

(4) binding of the substrate to humic acids [1].

It is interesting to note that enzyme adsorbed on goethite

displayed relatively higher specific activity than those on fine

organic clay, coarse clays and kaolinite. This implied that

adsorption by ligandexchange mayhaveno significant influence

on enzyme activity. Another explanation may be that the forma-

tion of enzymeiron complexes mayenhance itsbinding with the

substrate and resulted in more enzymatic products. Gianfreda et

al. [28] demonstrated that the activities of tannateurease sus-

pensions were greater in the presence of soluble iron species or

OHAl polymers. A large recovery of activity was observed forthe insoluble complexes obtained in the presence of ions. They

proposed that Fe3+ ions and OHAl polymers facilitated the

flocculation of tannateurease complexes and the formation of

more active tannatemetalurease complexes. The higher resid-

ual activities of fine clays may also be related to their higher

content of iron oxides.

3.4. pH-activity profiles

The pH-activity profile of free and immobilized enzyme is

shown in Fig. 3. In the range of pH 4.06.0, enzyme displayed

the highest activity at pH 5.5. A similar profile was observedfor the free and immobilized enzyme. No obvious shift for the

optimal pH of immobilized enzyme was observed as described

previously by some investigators [13].

As pH increased from 5.5 to 6.0, the activity of free

enzyme decreased by 71%, fine soil clays declined 4852%,

goethite decreased 45%, kaolinite 42% and coarse soil clay

decreased only 2437%. These results suggest that free enzyme

Fig. 3. Activities of free and immobilized acid phosphatase vs. pH.

Fig. 4. Activities of free and immobilized acid phosphatase vs. temperature.

is more sensitive than soil clay or mineral-bound enzymes to pH

changes. Moreover, it seems that enzymes on coarse clays were

more resistant to pH change than those on fine clays.

3.5. Effect of temperature

Fig. 4 outlines the activities of free and immobilized enzymes

at temperatures from 15 to 85 C. The figure clearly shows that

both free and immobilized enzymes had an optimal activity at

35 C. The enzyme, free or immobilized, was denaturated at

80 C. As temperature increased from 35 to 65 C, the activity

of free enzyme decreased by 87.5%, enzymes on fine clays and

goethite decreased by 73.277.4%. Coarse clays and kaolinite

declined by 67.171.8%.

Higher thermal stabilities of the organic clayenzyme com-

plexes were observed. This is attributed to the protective effect

of organic substances. Similar results were reported by Rao et

al. [13] who found that phosphates immobilized on organo-

mineral supports like OHAltannic acid and OHAltannic

acidmontmorillonite was more stable than free in solution. In

contrast, the enzyme boundon OHAlmontmorillonite showed

a higher sensitivity to thermal deactivation. Enzyme on kaolinite

displayed higher thermal stability than fine clays and goethite.

This may be ascribed to the tight binding of enzyme molecules

on the mineral. Our desorption data showed that most of the

enzymes on kaolinite could not be released by NaCl and phos-

phate. The relatively higher stability of coarse clayenzyme

complexes than fine clay complexes may be because that coarse

clay contains greater amounts of kaolinite.

4. Conclusions

Higher adsorption amounts and low percentage of desorption

of acid phosphatase were found on fine soil clays. More enzyme

molecules were adsorbed on soil clays in the presence of organic

components. However,enzymes on organic clayswere more eas-

ily released. One-third of the enzyme on goethite was adsorbed

via ligand exchange process. The majority of enzyme on kaoli-

nite cannot be easily removed. The activity of enzyme bound on

soil clays was inhibited and the thermal stability was increased

the presence of organic matter. Data obtained in this study are of


6/6


fundamental for a better understanding of enzyme stabilization

and the subsequent catalytic process in soil environments.

Acknowledgements

Thanks are given to Miss Lingyun Wei for technical assis-

tances in experiments. The research was financially supported

by the National Natural Science Foundation of China (Project

No. 40271064) and the International Foundation for Science

(C/2527-2).

References

[1] P. Ruggiero, J. Dec, J.-M. Bollag, in: G. Stotzky, J.-M. Bollag (Eds.),

Soil Biochemistry, vol. 9, Marcel Dekker, New York, 1996.

[2] J.M. Sarkar, A. Leonowicz, J.-M. Bollag, Soil Biol. Biochem. 21 (1989)

223.

[3] R.G. Burns, in: P.M. Huang, M. Schnitzer (Eds.), Interactions of Soil

Minerals with Natural Organics and Microbes, Soil Science Society of

America, Madison, WI, 1986.

[4] P. Nannipieri, P. Sequi, P. Fusi, in: A. Piccolo (Ed.), Humic Substancesin Terrestrial Ecosystems, Elesevier, Amsterdam, 1996.

[5] M.-C. Marx, E. Kandeler, M. Wooda, N. Wermbterc, S.C. Jarvis, Soil

Biol. Biochem. 37 (2005) 35.

[6] L. Gianfreda, J.-M. Bollag, in: G. Stotzky, J.-M. Bollag (Eds.), Soil

Biochemistry, Vol.9, Marcel Dekker Inc., 1996.

[7] Q. Huang, H. Shindo, Soil Biol. Biochem. 32 (2000) 1885.

[8] P. Fusi, G.G. Ristori, L. Calamai, G. Stotzky, Soil Biol. Biochem. 21

(1989) 911.

[9] L. Gianfreda, M.A. Rao, A. Violante, Soil Biol. Biochem. 23 (1991)

581587.

[10] L. Gianfreda, M.A. Rao, A. Violante, Soil Biol. Biochem. 24 (1992) 51.

[11] L. Gianfreda, M.A. Rao, A. Violante, Soil Biol. Biochem. 25 (1993)

671.

[12] M.A. Rao, L. Gianfreda, F. Palmiero, A. Violante, Soil Sci. 161 (1996)

751.

[13] M.A. Rao, A. Violante, L. Gianfreda, Soil Biol. Biochem. 32 (2000)

1007.

[14] B. Kelleher, A.J. Simpson, K.O. Willeford, M.J. Simpson, R. Stout, A.

Rafferty, W.L. Kingery, Biogeochemistry 71 (2004) 285.[15] Q. Huang, H. Shindo, T.B. Goh, Soil Sci. 159 (1995) 271.

[16] Q. Huang, M. Jiang, X. Li, in: J. Berthelin, P.M. Huang, J.-M. Bollag,

F. Andreux (Eds.), Effect of Mineral-OrganicMicroorganism Interac-

tions on Soil and Freshwater Environments, Kluwer Academic/Plenum

Publishers, New York, 1999.

[17] H. Shindo, D. Watanabe, T. Onaga, M. Urakawa, O. Nakahara, Q.

Huang, Soil Sci. Plant Nutr. 48 (2002) 763.

[18] S.A. Boyd, M.M. Mortland, in: J.-M. Bollag, G. Stotzky (Eds.), Soil

Biochemistry, Marcel Dekker Inc., 1990.

[19] A. Naidja, P.M. Huang, J.-M. Bollag, J. Environ. Qual. 29 (2000)

677.

[20] R.J. Atkinson, A.M. Posner, J.P. Quirk, J. Phys. Chem. 71 (1967) 550.

[21] Y. Xiong, Soil Colloids, vol. 2, Science Press, Beijing, 1983.

[22] C.H. Giles, T.H. MacEwan, S.N. Nakhwa, D. Smith, J. Chem. Soc. 56

(1960) 3973.

[23] Q. Huang, Z. Zhao, W. Chen, Chemosphere 52 (2003) 571.

[24] R.J. Sepelyak, J.R. Feldkamp, E.M. Timothy, L.W. Joe, L.H. Stanley, J.

Pharm. Sci. 73 (1984) 1514.

[25] H. Quiquampoix, in: J.-M. Bollag, G. Stotzky (Eds.), Soil Biochemistry,

Marcel Dekker Inc., New York, 2000.

[26] A. Naidja, P.M. Huang, Appl. Clay Sci. 9 (1994) 265.

[27] W.A. Dick, N.G. Juma, M.A. Tabatabai, Soil Sci. 136 (1983) 19.

[28] L. Gianfreda, M.A. Rao, A. Violante, Soil Sci. Soc. Am. J. 59 (1995)

805.

Documents

Huang - adsorção e dessorção de fosfatase