Immobilization of dextranase on bentonite

I m m o b i l i z a t i o n of d e x t r a n a s e on bentonite Madhu and K. A. Prabhu Biochemistry Division, National Sugar Institute, Kanpur 208017, India

(Received 23 May 1984; revised 2 October 1984) Dextranase (1,6-Ot-D-glucan 6-glucanohydrolase, EC3.2.1.11) from Penicillium aculeatum culture has been immobilized on a bentonite support. The matrix-bound e n z y m e could be stored as acetone-dried powder or as a suspension in acetate buffer, pH 5.6, f o r about three weeks at 4 n c without any loss o f activity. There was no change in the specific activity o f the e n z y m e on immobilization and the e n z y m e yield was 0 . 1 - 0 . 6 mg/g bentonite matrix. In the presence o f sucrose, thermal stability o f the immobilized e n z y m e was high and the bound e n z y m e could be used f o r about six cycles. Keywords: Enzyme; dextranase; immobilization; bentonite; Penicillium aculeatum

Introduction Several inorganic clarifying agents, such as Kieselguhr, charcoal, bentonite, Hyflo-Supercel and super-phosphate, are used in the sugar industry for the clarification I of cane juices. The application of dextranase 2 (1,6-a-o-glucan 6-glucanohydrolase, EC3.2.1.11) as a clarifying agent in the removal of dextran from cane juice is gaining importance in the sugar industry. Organic and inorganic supports have been employed in the immobilization of dextranase by several workers, a-7 This paper describes the study of properties of bentonitebound dextranase for use as a stable clarifying agent for cane juices.

Materials and methods Bentonite from Sigma, and CNCIa, SOCI2 and glutaraldehyde from BDH were used. D e x tranase

The enzyme used in these studies was isolated from NSI-4 culture as described previously.8 The enzyme was purified by cold acetone fractionation (66%), dissolved in acetate buffer, pH 5.6, and dialysed. The enzyme solution containing 200 units m1-1 (0.2 mg protein) was stored at 4°C with a few drops of toluene.

Penicillium aculeatum

Soluble dextranase assay

The assay was performed according to the method of Kosaric et al. as described previously,a using 2 ~tg enzyme protein in acetate buffer, pH 5.6, and incubating at 40°C for 20 min. Immobilized

dextranase assay

As in the study of free/soluble dextranase, immobilized/ insoluble dextranase was assayed by incubating 1 ml 0141 --0229/85/060279--04 $03.00 © 1985 Butterworth & Co. (Publishers) Ltd

bentonite suspension (2/ag bound protein/8 mg solids) and 50 mg dextran substrate in acetate buffer in a final volume of 3 ml at 40°C for 20 min, with shaking. Reducing sugars released were estimated as o-glucose by the DNS method and results are expressed in units (gmol ml -I min -l) and mg reducing sugar released in the reaction mixture in 20 min. Protein was estimated by the Lowry method. 9 Immobilization

o f d e x tranase o n b e n t o n i t e

In the preliminary studies on enzyme purification, it was found that dextranase was adsorbed on bentonite at pH 5.6 and was not eluted. Further experiments were carried out to optimize the conditions for insolubilization. Bentonite as a support was used after treating with cyanuric chloride, thionyl chloride 7 and glutaraldehyde. 1° Fifty mg quantities of the above supports as well as untreated original bentonite were stirred with 1 ml enzyme (0.2 mg) at pH 5.6 overnight a~ 4°C. The supports were removed by centrifuging in the cold and they were washed twice with pH 3.6 and pH 4.1, 0.05M to 0.1M acetate buffers. The supernatant and the washings were analysed for protein and activity. Activity remaining on the support or adsorbed was calculated from the difference in activities before and after treatment of the enzyme. To determine the optimum enzyme-to-carrier ratio in the adsorption/immobilization process, various amounts (50 to 1000mg) of bentonite were stirred with 1 mt dextranase enzyme in acetate buffer, pH 5.6, at 4°C for 24 h with continuous shaking. The samples were washed twice with acetate buffers of pH 3.6, 4.1 and 5.6, first at 0.05r~ and then 0.1 ra concentration, respectively, by suspending in buffer at 4°C for 20 rain and then centrifuging. Finally, the sample was suspended in the same buffer (pH 5.6) and stored at 4°C for about three weeks for enzymatic studies. A portion was also treated with cold acetone and the dry powder was stored at 4°C. The immobilized enzyme was checked by reusing the support matrix for enzyme activity as described above.

Enzyme Microb. Technol., 1985, vol. 7, June

279

Papers After the reaction, it was centrifuged and washed with buffer and used for the next cycle. To study the properties of immobilized dextranase, the reaction mixture was incubated at different temperatures and pH and with different chemical agents. Thermal stability of the free and immobilized enzyme was studied with and without sucrose (20%) at pH 5.6 and 50°C for different times and then taken for enzyme assay.

c E O v x E

Results a n d discussion Bentonite was selected as the support for dextranase immobilization because of its frequent use in the clarification of juices, its inert microporous structure with a large surface area and its high stability. No work seems to have been done on the application of immobilized dextranase in sugar-cane juices. Bentonite support in the original and glutaraldehydeactivated forms gave good results in immobilization. In both cases, the bound protein and activity on the support was about 50% of the original level. Bentonite treated with cyanuric chloride and thionyl chloride showed a retention of 55 and 41%, respectively, of the original activity (Table 1) and the bound protein on the support was 50 and 30%, respectively. The supports lost their adsorbed activity in subsequent cycles. Untreated and glutaraldehyde treated bentonite supports upon immobilization retained the full activity (protein basis) and the original homogeneous nature in suspension. The bound enzyme suspension could be preserved for more than two weeks at 4°C. Therefore, the glutaraldehyde-treated/untreated bentonite supports were used in all the enzyme studies. Enzyme yield on the bentonite support was 0.1 to 0.6 mgg -1 (Table 2). Bentonite-immobilized dextranase showed a sharp pH optimum at pH 5.6, as in the case of free bacterial dextranase, s in contrast to its soluble enzyme form, which exhibited a wide optimum pH range of 3 to

Table 1

Immobilization o f dextranase on bentonite support ( 1 0 0 m 9 ) . Dextranase added corresponded to an activity o f 200 units (0.2 mg protein) Dextranase bound

Treated bentonite Control (untreated) Cyanuric chloride T h i o n y l chloride Glutaraldehyde

Protein (mg}

Activity (units)

Activity retained (%)

0.090

98.6

49.3

0.090 0.065 0.090

109.8 83.3 98.9

54.8 41.6 49.8

/

o

rr

0

I 2

,

0

,

I 4

6

8

pH

Figure1 Effect o f pH e, Free; x, immobilized

on

free

and

immobilized

dextranase:

5.8 (Figure 1). This may be due to loss of some basic groups during immobilization on bentonite. The Michaelis constant, Kin, and Vmax calculated from a Lineweaver-Burk plot (Figure 2), for the free and immobilized enzyme were found to be altered. A decrease in Vmax and an increase in Km values can be explained on the basis that the enzyme is immobilized not only on the surface but also within the bentonite particles. Similar results have been reported by Weetall et aL11 The temperature optimum was unchanged after immobilization of dextranase but heat stability was reduced (Figure 3). The activity was retained at 45°C for only 30 rain whereas the soluble enzyme retained activity at 50°C for 60 min. Sucrose at a concentration of 20% had a favourable effect on restoring the thermal stability of enzyme in the immobilized form (Figure 3). Sucrose, due to its highly polar nature, protected the enzyme from thermal denaturation. The change of reaction rate of the enzymes can be seen clearly on the Arrhenius plot (Figure 4) for which bound and free enzyme activities give straight lines. In the bound enzyme, the gradient changed abruptly on reaching 50°C. It can be concluded from Figures 3 and 4 that the activity loss was due to thermal deactivation of enzyme. Table 3 compares data for the kinetic parameters of both soluble and immobilized dextranase. Matrix-bound dextranase (0.25 m g g -1 support) was selected because

Table 2 Effect of bentonite (carrier) on enzyme yield Thermal stability (rain)

Dextranase

Bentonite

enzyme (~tg protein)

(carrier) (rag)

Enzyme yield (rag g-l)

(% bound protein)

No sucrose (45 ° C)

With 20% sucrose (50 ° C}

Repeated use o f bound protein (approx) cycles

200 2O0 200 200 200

50 100 200 400 1000

0.6 0.5 0.5 0.25 0.1

50 75 100 100 100

10 10 20 30 20

N.t. N.t. N.t. 60 N.t.

1 2 2 6 5

Activity

N.t., Not tested

280

Enzyme Microb. Technol., 1985, vol. 7, June

/mmobilization of dextranase on bentonite: Madhu and K. A. Prabhu 180 140

6O 20 20

1.0

0

1.0

-I/Km Figure 2 Lineweaver--Burk bilized

2.0

30

4.0

5.0

EO

I/S ( % dextran-') plot of dextranase: e, Free; x, immo-

the heat tolerance, cycle time and activity (Table 1)in the reuse of enzyme were greater at 0.25 mg g-1 than at 0.5 and 0.6 mg g-~. The slight excess of bentonite in bound protein at 0.25 mg g-1 yield possibly protected the bound, enzyme from leakage. Dextranase immobilized on bentonite could be preserved in a dry form on treatment with acetone. In buffer suspension or sucrose suspension it was stable for a b o u t three weeks at 4°C. Similar results have been reported for immobilized bacterial dextranase. 3,s The bound enzyme could be used repeatedly for about six cycles (Figure 5) without any appreciable loss of activity. In the eighth cycle ~50% of the original activity was lost, as in glucose isomerase of heat-fixed cells ofLactobaeillus sp.12 In the enzyme reaction studies with inhibitors it was found that bound enzyme was less inhibited in the presence of Pb 2+ and Cu 2+ ions than the free form. Activity could be regained by washing the support (Table 4). The bentonite support possibly acted as an adsorbent for these metallic inhibitors and thus lowered the inhibition of bound enzyme. Such an effect has also been reported in polystyrene-bound fl-D-fructofuranosidase. 13 Chelating agents 4-chloromercuribenzoate (4CMB), EDTA, mercaptoethanol, sodium ascorbate, sodium azide and potassium ferricyanide inhibited matrix-bound enzyme but not its free soluble form, indicating the release of free active disulphide groups during immobilization on bentonite. In the present study, no loss in specific activity on immobilization was observed, as reported by other workers. 3,s The activity retained and cycle times obtained were very high, although the enzyme showed less thermal

stability. The decreased thermal tolerance of bentonitebound dextranase is unclear. According to Chibata z4 the physical adsorption process is not suitable for obtaining highly thermostable immobilization systems. The amount of protein bound per gram of matrix was quite low compared with previously reported results, a's Thus, enzyme overcrowding as a factor affecting the relative specific activities is not likely to be significant. Bentonite-bound dextranase was found to be quite stable. The ease and economy of the active insoluble preparations of dextranase indicate that it is the best of the methods. Application of this bentonite matrix-bound enzyme in acetone powder form or suspension in acetate buffer or sucrose solution will help greatly in the sugar industry, with the possibility of continuous removal of dextran from juices. The bound enzyme was stable and in a recoverable

/

I00

80

o~

60

40

2O , 0

I 2O

,

I 40

,

I 6O

Temperature (°C) Figure 3 Thermal stability of dextranase: e, Free + sucrose (20%) for 60 rain; o, immobilized + sucrose (20%) for 60 rain; ~, immobilized for 30 min

Table 3 Kinetic parameters of free and immobilized dextranase Parameters Adsorption/immobilization (%) Enzyme yield

Free (soluble)

R

Loss of activity (%) Specific activity

Krn Vmax (lzmol m1-1 min - l ) Optimum pH Optimum temperature (°C) Thermal stability Thermal stability with 20% sucrose Stability

1000 units mg -1 protein 55.5 × 10 -2 2.50 3.0 to 5.8 50 50°C f o r 60 min 50°C for 60 min Retained 100% activity at 4°C in acetate buffer (pH 5.6) for 90 days

Immobilized (insoluble) 50 0.25 mg g-1 matrix 250 units/g Nil 1000 units/rag protein 71.4 X 10 -2 1.56 5.6 5O 45°C for 20 min 50°C for 60 min Retains 100% activity at 4°C for 1 5 - 2 0 days in acetate buffer suspension (pH 5.6) or in 20% sucrose suspension. Acetonetreated enzyme-bound matrix highly stable. Retains activity for about six cycles, 1 g 1-1 bound enzyme support, removed 60% dextran from cane juice at 50°C in 1 h

E n z y m e M i c r o b . T e c h n o l . , 1985, vol. 7, J u n e

281

Table 4

form. Preliminary data on removal of dextran from cane juice using bound dextranase support (1 g 1-l) showed 60% removal of dextran at 5O’C in one hour.

Effect

and immobilized

of some inorganic

Remaining Chemical agents (6 X lo-” M)

0.0 [

salts and chelating

I

agents on free

dextranase activity

(%)

Free

Immobilized

HgCf, HgCf,

100.0 0 N.t.

100.0 0 0e

93.5

31.5

Lead acetate Lead acetate CuSO, .5H,O ‘&SO,. 5H,O EDTA Potassium ferricyanide Sodium ascorbate lodoacetate 4CMB (4chloromercuribenzoate) Mercaptoethanol NaN,

0 N.t. 0 N.t. 100.0 100.0 100.0 100.0 0 100.0 100.0

0 50= 0 50a

Control

FeSO,. JH,O

75.0 71.0 50.0 50.0 50.0 60.5 76.0

aAfter two washings with buffer N.t., Not tested

0.0

2.7

I

I

I

I

3.0

3.1

3.2

33

IO3 x I/T’(K-‘1 Figure 4 Effect

of

temperature

on

dextranase

(Arrhenius

plot):

l, Free; x, immobilized

1 2 3 4 5 6 I 8 9 10 11 12 2

d-

4

6

8

Cycle number Figure 5 Repeated

282

use of dextranase

(batch process) immobilized

Enzyme Microb. Technol., 1985, vol. 7, June

13 14

Meade, G. P. and Chen, J. C. P. Cane Sugar Handbook 10th ed., John Wiley and Sons, New York, 1977, p. 183 Madhu, Shukla, G. L. and Prabhu, K. A. ht. Sugar J. 1984, 86 (1025), 36 Kennedy, J. F. and Kay, M. Carbohydr. Rex 1977, 59, 55% 561 Gray, C. J. and Livingstone, C. M. Biotechnol. Eioeng. 1977, 19,349-364 Ramesh, V. and Singh, C. Biochem. Biophys. Rex Commun. 1980,97,179-786 Cheetham, N. W. H. and Richards, G. N. Carbohydr. Res. 1973,30,99-107 Monsan, P. and Durant, G. FEBS Lett. 1971, 16, 39 Madhu and Prabhu, K. A. Enzyme Microb. Technol. 1984, 6, 217-220 Lowry, 0. H., Rosebrough, H. J., Fax, A. L. and Randall, R. J.J. Biol, Chem. 1951, 193, 265 Chibata, I., Tosa, T. and Sato, T. Appl. Microbial. 1974, 27,878 Weetall, H. H. and Hersh, L. S. Biochim. Biophys. Acra 1970, 206,54-60 Bhatia, M. and Prabhu, K. A. BiotechnoZ. Bioeng. 1980, 22, 1957 Filippusson, H. and Hornby, W. E. Biochem. J. 1970, 120, 215 Chibata, I. Immobilized Enzymes John Wiley and Sons, New York, 1978, p. 134