Immobilization of enzymes on clays and soils

Soil Bid. B&hem.

Vol. 21. No. 2. pp. X3-230.

1989

0038-0717;8953.00 + 0.00 Copyright C 1989 PergamonPressplc

Printed in Great Britain. All rightsreserved

IMMOBILIZATION

Laboratory

OF ENZYMES AND SOILS

ON CLAYS

JAWED M. SARKAR, ANDRZEJ LEONOWICZ and JEAN-MARC BOLLAG’ of Soil Biochemistry. Department of Agronomy, The Pennsylvania State University, University Park, PA 16802, U.S.A. (Accep~d IO October 1988)

Summary-Various enzymes such as glucose oxidase. j?-D-ghrcosidase,acid phosphatase, tyrosinase and laccases were immobilized on clays and soils activated with 3-aminopropyltriethoxysilane and glutaraldehyde. After immobilization the enzymes retained a large amount of their original activities. The retention of lactase activity increased with the increase of clay content in soils, whereas the activity of g-D-glucosidase, tyrosinase and acid phosphatase decreased. The immobilized enzymes showed varying degrees of resistance to proteolysis and storage at high temperatures. After addition to soil suspensions, soluble lactase and glucose oxidase were rapidly inactivated (100 and 85% loss of activity in I5 days for lactase and glucose oxidase, respectively) whereas after immobilization, these enzymes were extraordinarily stable (I2 and 25% loss of activity in I5 days for lactase and glucose oxidase. respectively). The possibility of incorporating the stabilized enzymes into soil for the improvement of numerous desired biochemical processesis discussed.

INTRODUCTION

in soil catalyze numerous important reactions responsible for maintaining the biological activity of soil. The activities of certain enzymes in soil have been correlated with plant growth and are thought by some researchers to serve as useful indices of soil fertility. Examples of such enzymes include carbohydrases which take part in the carbon cycle, soil aggregation (Kiss et al., 1978) and in the transformation of cellulosic materials (Sarkar and Burns, 1984); proteinases, amidases and deaminases which function in the nitrogen cycle (Ladd and Paul, 1973); and oxidoreductases which participate in the humification process (Flaig, 1955) and in the coupling of certain xenobiotics to humic materials (Bollag. 1983). Upon their release from microbial cells these soil enzymes become physically absorbed onto soil colloids or covalently bound to soil organic matter. The physically adsorbed enzymes in soil are readily inactivated due to adverse soil conditions, while the enzymes bound covalently to humic colloids persist in soil and perform their important functions. Thus soil provides a natural system where enzymes become immobilized to clay and to humus (McLaren, 1975). As Burns (1986) stated, it is difficult to assess clearly the biological significance of the immobilized enzymes in soil, but it seems that their biochemical activity is more important for certain reactions than that of the viable microbial cells. Often it occurs that the total microbial numbers or specific groups of microorganisms are poorly correlated to the observed biochemical reactions. An alternative approach to understanding the relationship between immobihzed soil enzymes and their humic acids was used,by Rowell et al. (1973) who prepared p-benzoquihone-pronase, and pEnzymes

*Author

to whom correspondence should be addressed.

benzoquinone-trypsin copolymers, as analogues of humic acid-enzyme complexes. They found that although the activity of immobilized enzyme was considerably reduced, they displayed greater thermostability. Sarkar and Burns (1984) synthesized p-D-glucosidase and cellulase-phenolic copolymers and found that these stable enzyme-phenolic copolymers were similar to the naturally-occurring soil enzyme-humic complexes. The immobilization of enzymes on synthetic or natural humic materials often causes inhibitory effects to the enzymes. For example, oxidoreductases (Pflug, 1980; Sarkar and Bollag, 1987), protease (Ladd and Butler, 1969). invertase and phosphatase (Malcolm and Vaughan, 1979a, b) were all inhibited by humic materials. The losses occurring to enzymes by their immobilization to humic materials lead us to search for an alternative nonbiodegradable natural support without the loss of much of their original activity. This paper describes the immobilization of oxidoreductases and hydrolases on soils, bentonite and kaolinite and examines the stability and the reusability of the immobilized enzymes. .MATERIALS AtiD METHODS

Soils

Freshly collected silt loam soil (pH 6.4; silt 59%; sand 17%; clay 24%; and organic matter 3.2%) and sandy loam soil (pH 4.0; silt 18%; sand 72%; clay 10%; and organic matter 2.3%) were sieved (< I mm), ground to a fine powder and stored at room temperature. Bentonite and kaolinite powder were used as supplied (Ward’s Natural Science Establishment, Rochester, N.Y.). Source of enzymes

Laccases (p-diphenol oxidase) (EC 1.10.3.2) were prepared from strains of the basidiomycetes Trametes cersicolor and Rhkoctonia praticola as described by

223

JAWED

224

M.

SARKAR et al.

Bollag and Leonowicz (1984). Glucose oxidase (EC I. I .3.4). tyrosinase (EC I. 14.18. I). /?-D-glucosidase (EC 3.2.1.2 I). protease (Streptom_~es cuespirosus), invertase (EC 3.2.1.16) and acid phosphatase (EC 3. I .3.2) were purchased from Sigma Chemical Co., St Louis, MO. Immobikation

ofenzymes

Enzymes were immobilized on clays and soils using the techniques described for the activation of porous glass by Robinson et ai. (1971). We used the modified procedure: I g of the soils. bentonite or kaolinite were treated with cone. HNO, with boiling and continuous stirring for 1 h. HNO, was removed by centrifugation (I 3,000g) and the pellets were washed several times with distilled water until the pH of the supernatants reached 6.0. The supports were then activated by immersion in a 2% solution of 3-aminopropylt~ethoxysilane (APTES) in acetone. The activated supports obtained were then treated with 5 ml of 5% glutaraldehyde prepared in 100 rnM phosphate buffer (pH 7.0). The mixture was then evacuated for I h to remove excess glutaraldehyde and the pellets were washed several times with deionized water and then with 100 mM phosphate buffer (pH 7.0). These supports were then used for the immobili~dtion of various enzymes. Equal amounts of the various enzymes (proteins) were solubilized separately in 2.0ml of 100 mM phosphate buffer (pH 7.0) and stirred with the treated supports at 4’C for 34 h. The enzyme supports were centrifuged. washed thoroughly with water until no further activities were detected in the washings, and stored at 4’C as moist cakes. rWcwswcnrmtof en:yme activities Laccases, tyrosinase and glucose oxidase activities were estimated using the following substrates at the final concentrations of IO mM 2,6-dimethoxyphenol, 0.5 rnbf 3,4-dihydroxyphenylaIanine and 30 mM glucose, respectively. The activities of these enzymes were measured polarogrdphically using a biological oxygen monitor (Model 5300 Yellow Springs Instrument Co., Ohio) equipped with a calibrated Clark oxygen electrode and a linear recorder (Varian Model 20). Then 3.0 ml of a lOOmu citrate phosphate buffer, pH 3.3 for a taccase of 1: cersieolot and pH 7.0 for a lactase of R. praticofa. tyrosinase and glucose oxidase, were saturated with oxygen and the desired amount of enzymes or substrates were injected through a side port of the electrode. Vigorous agitation was achieved using a Teflon-coated magnetic stirring bar. One unit of an enzyme was defined as that amount which caused 1 pmol min-’ oxygen to be consumed in a 3.0 ml reaction mixture at 2.X and optimum pH. p-D-Glucosidase and acid phosphatase activities using p-nitrophenyl were measured P-D-glucopyranoside (Sarkar and Burns, 1984) and p-nitrophenyl phosphate disodium (SIGMA 104R phosphate substrate; Batistic et al., 1980), respectively, as substrates. In both cases release of p-nitrophenol was measured and enzyme units were defined as p-nitrophenol release min-’ and mg-’ of dry wt. The specific units of each enzyme were calculated by dividing the units per milligram of protein. The protein concentration was determined by the method of Bradford (1976) using the Bio-Rad

protein reagent (Bio-Rad Lab., Richmond, Calif.) with bovine serum albumin as standard. Eflect of temperature. The resistance to elevated temperatures of free and immobilized lactase, ty rosinase, glucose oxidase and ~-~gIucosidase was examined. Free and immobifized enzymes were incubated at various temperatures for 60min prior to measurement of their residual activities. Efict of protease. The effect of protease (from Streptomyces cuespitosus, Sigma Chemical Co., St Louis, MO.) on free and immmobiliz~ lactase, tyrosinase, glucose oxidase and acid phosphatase was examined. About 50 units of free and immobilized enzymes were allowed to react separately with protease (22 U, 6 mg) in the presence of 50 mM Tris-HCI buffer (pH 8.0) at 37’C in a shaking water bath for I2 h. Samples of 100 ~1 were periodically withdrawn and the reaction terminated by rapidly reducing the temperature. The pH of the reaction mixture was adjusted using the appropriate buffers required and the residual activity of each enzyme was measured as described above. The residual protease activity was also measured using casein as a substrate (Ladd and Butler. 1972). Stability in soil suspensjo~. The stability of free and immobilized lactase and glucose oxidase was examined by incorporating equal units of these enzyme preparations into various amounts of freshly collected silt loam soil. The amount of soil ranged from 5 to 180 mg ml-’ water. These samples were incubated at 25-C for 30 days. At various times during this period, samples were withdrawn and the residual activities of the free and immobilized enzymes determined. Reusabi1it.vof immobilized lactase. The reusability of the immobilized lactase was examined by packing a small glass column with 100 mg (wet wt) of silt loam soil immobilized faccase containing about 20 pg protein. The column was equilibrated and eluated at a flow rate of IOml min-’ with IOOmM citratephosphate buffer (pH 3.6), and lactase activity was determined in the eluted solution. Once the column eluate was colorless, 0.1 mM 2,6-dimethoxyphenol dissolved in the same buffer was passed through the column; the absorbance of the collected fractions was measured at 468 nm to determine the remaining lactase activity. RESL’LTS

Enzymes were successfully immobilized on soils, bentonite and kaolinite provided they had been treated with concentrated HNO, and activated by 3-aminopropyltriethoxysiIane (APTES) and glutaratdehyde. As an example, when a silt loam soil was pretreated with these chemicals in sequential order prior to the addition of a lactase from T. uersicolor, about 95% of the lactase protein was immobilized with a retention of 83% of the originally applied enzyme activity (Fig. I). However, when the silt loam soil was simply mixed with lactase in the buffer presence of IOOmM sodium phosphate (pH 7.0), only 17% of the enzyme protein was immobilized with a 5% retention of enzyme activity (Fig. I). We extended our investigation to other enzymes

Immobilization of enzymes on clays and soils 100 -

3

N

i iij 0

50-

3

GA

Fig. 1. Immobilization

of &case

APTES

APTES GA

y

BENTONITE

HNO3 APTES

HNO3 APTES GA

KAOLINITE

APIES:

rately concerning determination of retainment of its activity on a suitable support. Indeed, it is very difficult to ascertain the component in soil which favors the attachment of an enzyme. For instance, we were interested to know why a silt loam soil retained a higher lactase activity than a sandy loam soil and investigated this question by comparing a number of well-defined Pennsylvania soils containing various amounts of clay, sand and silt. Foilowing their activation, equal units of a Trutnetes Iaccase were immobilized to a known amount of each soil. The results of this experiment showed a direct linear relationship between the retention of lactase activity and the amount of clay present in the soil (Fig. 3), indicating that clay particles are the major support of an active immobilized lactase. In a separate experiment we further investigated this phenomenon by adding various amounts of bentonite clay (ranging from 2.5 to 100%) to pure sand. These clay-sand mixtures were then used for the immobilization of various enzymes. As demon-

TYROSINASE

m

HN03 GA

of 7: wsifolor on a silt loam soil. GA: glutaraldehyde. 3-aminopropyltriethoxysilane.

and examined the immobilization of tyrosinase, glucose oxidase, protease, invertase, acid phosphatase and B-D-glucosidase on soils, bentonite and kaolinite. Figure 2 presents the results from immobili~tion of a taccase from T. t-ersicolot, a tyrosinase, an acid phosphatase and a P-D-glucosidase to bentonite, kaolinite, sandy loam and silt loam soil. Generally it could be determined that all protein was immobilized on the tested supports, but there was considerable variation in the retained activity of the different enzymes. While almost all activity was retained on bentonite for the Tmmetes lactase, only about 75, 60 and 17% was recovered on bentonite for tyrosinase, fl-o-glucosidase and acid phosphatase, respectively. There also was considerable difference in the remaining activity of the enzymes on the various supports. For instance, tyrosinase was equally retained on bentonite, kaolinite, sandy loam and silt loam soil, but recovery of phosphatase activity on bentonite was much less than on silt loam soil. All these results show that each enzyme has to be investigated sepa-

LACCASE ( T. versicolorI

HN03

ACID PHOSPHATASE

0

SANDY LOAM SOIL

p-0-GLUCOSIDASE

m

SILT LOAM

Fig. 2. ImmobiIization of enzymes on varioq types of clays and soils.

SOIL

226

JAWED

M. SARKAR et al.

OOIS CLAY

(%I

Fig. 3. Etfect of the amount of clay in various types of Pennsylvania soils on the immobilization of T. cersicolor lactase.

in Fig. 4. the activity of the immobilized laccases of T. cersicolor and R. prabcola was increased almost linearly as the clay content of the sand increased. The activities of T. cersicolor and R. praticola retained on the various clay-sand mixtures were fairly similar. Contrary to the behavior of laccases on clay-sand mixtures, the amount of tyrosinase, acid phosphatase and fl-D-glucosidase activity immobilized on clay-sand mixtures was slightly decreased as the clay content in these mixtures increased. To optimize an enzyme immobilization procedure it is important to determine the capacity of the

BENTONITE

ADDED

(X)

Effect of bentonite added to sand on the immobilization of various enzymes.

strated

support for the greatest retention of enzyme activity. When various amounts of lactase were added to a constant weight of bentonite and a silt loam soil, the retained lactase activity increased linearly up to 20 U mg-’ of support (Fig. 5). No enzyme activity was released when supports were washed several times with l00m~ phosphate buffer (pH 7.0) and deionized water. When the amount of lactase exceeded 20 U mg-’ of support, enzyme activity ap peared in the supernatant, suggesting the saturation of the support.

SILT

LOAM

SOIL

/-I=--=-

LACCASE

AOOED

( S. U. / mg

WPPort )

Fig. 5. Addition of different amounts of 7: rersicolorlactase to silt loam soil and bentonite (S.U. = specific units).

Immobilization of enzymes on clays and soils Properties of free and immobilized en:ymes Effect of temperature. Incubation for 1 h of free and immobilized glucose oxidase, /I-o-glucosidase, lactase (of T. uersicolor) and tyrosinase, revealed differences in the resistance of these enzyme preparations to exposure at elevated temperatures (Fig. 6). For instance, at 6O’C free and immobilized lactase retained 25 and 50%, respectively, of their original activities; free tyrosinase lost all activity whereas the immobilized tyrosinase retained 40% of its activity; free and immobilized glucose oxidase retained 50 and 90%. respectively, of their original activities; free /I-o-glucosidase retained 50% of its original activity, whereas immobilized fl-o-ghicosidase retained 85% of its activity. In conclusion, immobilization of these enzymes on a silt loam soil rendered them more resistant to thermal degradation than their soluble counterparts. Eflct of protease. Free and immobilized enzymes were compared in their resistance to protease (Fig. 7). The residual activities of the free and immobilized enzymes-as well as protease-were periodically measured. There were no indications that protease was immobilized or absorbed on the treated clays and

(

LACCASE

,bo

l”

tGL"COSEOXIOASE

0

20

40

TEMPERATURE

TYR;NAXo

k!,

0

\

60 (*C

soils. In addition, proteolytic activity, determined in an assay with casein as substrate, did not change during incubation. Lactase. About 75% of the activity of free lactase was lost after a 5-h exposure to protease. and only 5% of its original activity remained after a 12-h exposure. In contrast. when immobilized lactase was incubated with protease under conditions identical to those of the free lactase. the immobilized lactase retained 65 and 37% of its original activity after 5 and 12 h. respectively. Tyrosinase. Both free and immobilized tyrosinase were susceptible to protease attack. However, the immobilized tyrosinase retained 15% of its original activity, whereas the activity of the free enzyme was undetectable after a 12-h incubation with protease. Glucose oxiduse. Free glucose oxidase seems to be quite resistant to protease digestion. However, an 8-h incubation of the free enzyme with protease resulted in a 20% loss of its original activity, whereas the immobilized enzyme showed no loss in activity. Acid phosphatase. Free acid phosphatase was extremely sensitive to protease, a l-h incubation resulting in 90% loss in activity, whereas the immobilized enzyme showed no loss in activity during the

o1

50

227

60 1

0

20

40

TEMPERATURE

60

80

1 lC 1

Fig. 6. The effect of a I-h exposure to various temperatures on the activity of various free and bentonite-immobilized enzymes.

0.0

Ol

3.0

GLUCOSE

6.0

9.0

OXIOASE

0.0

HOURS

12.0

3.0

AC10

6.0

9.0

PHOSPHATASE

Fig. 7. ERect of protease on the activity of various free and immobilized

a

LACCASE

enzymes.

12.0

F

0

FREE

45

SILT

135

160

LOAM

t

t

r

SOIL

(rng)

IMMOGILIZED

IMMOBILIZED

GLUCOSE

LACCASE

DAY

DAYS

DAYS

DAYS DAYS

DAYS

7 9 I5

DAYS

II

DAY

, 3

0 OAYS

OXIDASE

IS DAYS

DAYS 9 II

3 DAYS 7 DAYS

I

0 DAYS

. --.

_ _ _..

.-1

-

.

.

-.

_.

of free and immobilized luccase and glucose oxidase in varying amounts of silt loam soil.

90

LACCASE

._ _ -

Fig. 8. Stability

0

15

30

60

k

% m

L

yr

i?:

Immobilization

ELUATE

of enzymes on clays and soils

(11

Fig.

9. Effect of a continuous flow (IOmlmin-‘) of a 2,6-dimethoxyphenol solution (0.1 mM) through a column (IOcm length and Smm dia) containing silt loam soil immobilized T. rersicolor lactase on the remaining lactase activity.

same period. Furthermore, after a 3-h exposure, the free enzyme retained almost no activity, whereas the immobilized enzyme still possessed 60% of its original activity. Srobilir_r in soil suspensions. The stability of free and immobilized lactase and glucose oxidase preparations was assessed after incorporation into suspensions of various amounts of soil (Fig. 8). Free lactase began to lose its activity immediately after its incorporation into the soil suspension. This loss in activity depended upon the amount of soil and the duration of the incubation. No activity of the free lactase was found when incubated for IS days with 45 mg of soil. On the other hand, the immobilized lactase was remarkably stable, losing only 12% of its activity in a IS-day incubation with 45 mg of soil and only 25% with l80mg of soil (Fig. 8). Like the free lactase, the free glucose oxidase was also unstable in soil. After incubation with 45 mg of silt loam soil for a period of 15 days, only 15% of its original activity remained and no activity was found with 90 mg of soil. The immobilized glucose oxidase was more stable. Only a 25% loss in activity occurred after I5 days of incubation with 45 mg of soil and a 33% loss with 180mg of soil (Fig. 8). Reusabilit)

The possibility of reusing the lactase immobilized on silt loam soil in a continuous manner was also investigated. One hundred mg (wet wt) of immobilized lactase (2Opg protein) packed into a glass column effectively transformed the substrate 2,6-dimethoxyphenol when a solution of 2,6-dimethoxyphenol (0.1 mM) was continuously pumped through the column. The results indicated that the immobilized enzyme retained 25% of its original activity even after 41. of substrate solution had passed through the column (Fig. 9). DISCUSSION

The mechanism by which enzymes can be immobilized on clay minerals include cation exchange,

229

physical adsorption and ionic binding (Theng. 1979; Mortland. 1970). It is difficult to predict whether the immobilization of enzymes on these supports would decrease their activities since any involvement of the active site in binding to the support will reduce or eliminate activity. For instance, urease and glucose oxidase were immobilized by adsorption on hexadecyltrimethylammonium (HDTMA)-smectite with full retention of their activities (Boyd and Mortland. 1985; Garwood et al., 1983). However, under similar experimental conditions, horseradish peroxidase and arginase which were also strongly adsorbed on HDTMA-smectite exhibited no activity whatsoever. In this investigation a number of oxidoreductases and hydrolases were covalently linked to various types of soils and clays using APTES and glutaraldehyde as “coupling agents”. Using this technique almost 100% of the enzyme was immobilized on the supports and the majority of the enzymic activity was retained. Most activity was recovered for the lactase on all supports. whereas phosphatase was the least retained. Although it is difficult to explain these differences in the retention of enzyme activity, it may be assumed that the lactase, tyrosinase and /I-D-glucosidase active sites are in no way proximal to the binding sites, whereas the active site of acid phosphatase may be related to the binding site. It follows then that the immobilization of the protein is not inevitably related to the enzyme activity retained, since an enzyme protein may be strongly bound by the clay but display a complete absence of enzyme activity. Our results indicated that clay particles increased the immobilization of lactase activity but had little effect on the immobilization of acid phosphatase, /?-D-glucosidase and tyrosinase, since we found a greater retention of lactase activity in those soils with higher clay contents. In contrast to lactase, acid phosphatase activity retained decreased with increasing amounts of bentonite (hydrophilic clay). This result confirmed our finding that retention of acid phosphatase activity was less in bentonite than kaolinite and soils. The decreased retention of enzyme activity on clay may be attributed to either an inaccessibility of the active site to the substrate as a result of the manner in which the enzyme was adsorbed to clay or to some loss of intrir&enzyme activity upon adsorption to clays. There are numerous reports that the activities of several other enzymes were decreased upon adsorption to clay (Makboul and Ottow, 1979; Pflug, 1982). The stabilities of immobilized enzymes vs free enzymes towards proteolysis, at elevated temperatures and following incorporation into soil are of great interest. It is known that soil is an inhospitable environment for free extracellular enzymes since they are denatured, degraded or otherwise inactivated upon addition to soil (Burns, 1978). Enzymes immobilized on humus or clay colloids, on the other hand, are resistant to proteases and other denaturing compounds (Sarkar ef al., 1980; Nannipieri ef al., 1982). Stability of extracellular enzymes is essential for them to survive in soils long enough to degrade exogenous substrates. Unfortunately the indigenous extracellular enzymes or free enzymes added to soil are rapidly inactivated. Contrary to free enzymes the

JAWED M. SARKAR er al.

230

immobilized enzymes are very stable in soil and may act as catalysts for the degradation of substrates. In conclusion, the immobilization of enzymes on soil, bentonite and kaolinite provides an environment which resembles their own natural “status” in soil. Thus, it should be possible to incorporate the stabilized enzymes into soil with considerable retention of their activity. This procedure can thus be used for the catalysis of numerous biochemical processes.

Currently we are investigating the application of immobilized enzyme technology to the removal of xenobiotics from the terrestrial environment. Acknowledgements-This work was supported in part by a research grant from the U.S. Geological Survey and by the Pennsylvania Agricultural Experiment Station.

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jarkar J. M.. Batistic L. and Mayaudon J. (1980) Les hydrolases du sol et leur association avec les hydrates de carbone. Soil Biology & Biochemistry 12, 325-328. Sarkar J. M. and Burns R. G. (1984) Synthesis and properties of p-o-glucosidase-phenolic copolymers as analogues of soil humic-enzyme complexes. Soil Biology & Biochemisfry

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