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Noncovalent and reversible immobilization of chemically modified amyloglucosidase and beta-glucosidase an DEAE-cellulose
Process Biochemisny 29 (1994) 443-44X 0 1994 ElsevierScienceLimited
Printedin Great Britain.All tightsreserved O32-9592/94/$7.00
Noncovalent and Reversible Immobilization of Chemically Modified Amyloglucosidase and betaGlucosidase on DEAE-Cellulose Renu Tyagi & M. N. Gupta* Chemistry Department, (Received
Indian Institute of Technology,
14 June 1993; accepted
Delhi, New Delhi-1 10 016, India
8 July 1993)
The acylation of amino groups of proteins by pyromellitic dianhydtide (PMDA) leads to an increase in negative charges on the protein surface by 4 units for every amino group modified. This reaction has been used to mod@ enzymes for the pqvose of adsorption on DEAE-cellulose. The 49% amino groups of amyloglucoskiase were modijied with 90% residual enzyme activity. Whereas the native enzyme bound to DEAE-cellulose and could be eluted out with 0.1 M sodium chloride, the modeed enzyme exhibited stronger binding and could only be eluted with 0.25’ M sodium chloride. In the case of a&orbed native enzyme, heating at 60°C for 1 h led to desorption of 97% protein. In the case of the modified enzyme, the immobilized preparation retainedfull enzyme activity under similar conditions. beta-Glucosidase (which did not bind to DEAE-cellulose) upon modification with PMDA was found to bind to DEAE-cellulose and could be eluted out with 97% protein recovery by washing with O-2 M sodium chloride. Thus, chemical modification with PMDA may be a useful and general strategy for obtaining enzyme derivatives for reversible adriorption on anion exchangers.
INTRODUCTION
sufficient. Chemical modification of the protein to create appropriate charges on the surface is an obvious strategy which has been occasionally employed4J. Solomon and Levin4 used succinic anhydride to modify amyloglucosidase and the modified amyloglucosidase was bound to DEAEcellulose. Recently, the present authors have modified trypsin with both succinic anhydride and pyromellitic dianhydride (PMDA); the modified enzyme (Scheme 1), unlike the native one, bound to DEAE-cellulose. Pyromellitic dianhydride was found to be a better reagent for this purpose since for the same extent of chemical modification, the enzyme
Adsorption on ion exchangers constitutes a fairly successful approach to enzyme immobilization for industrial application’. The advantages of such noncovalent methods for protein/enzyme immobilization have been reviewed recently2+3. It is not always feasible to bind an enzyme on an ion exchanger with adequate strength as the surface charges on the enzyme may not be appropriate or
*Corresponding author: M. N. Gupta. Telephone: 9 1 11 666 979 ext.7615. Fax: 91 11686 2037.
443
444
R-NH2
Renu Tyagi, M. N. Gupta
Protein
RNH-CO
coo-
-0oc
coo-
o-
+
PMOA moditied deriwtii
PMOA
Scheme
1.
modified with PMDA retained significantly higher activity than the enzyme modified with succinic anhydride. In this work, the authors extend their approach to immobilize two biotechnologically useful enzymes ,viz. amyloglucosidase and betaglucosidase on DEAE-cellulose.
MATERIAL
AND METHODS
Amyloglucosidase (from Aspergillus n&r), beta- glucosidase, (from almonds), p-nitrophenyl alpha-n-glucopyranoside (NPG) and p-nitrophenyl beta-n-glucopyranoside (PNGP) were purchased from Sigma Chemical Co., USA. Pyromellitic dianhydride (PMDA) was a product of Eastman Kodak Co., USA (the authors thank Prof. A. M. Klibanov, MIT, USA for the gift of this chemical). All other reagents were of analytical grade. Enzyme Assay (i) Amyloglucosidase The enzyme assay was performed using NPG as substrate”. Each assay contained enzyme (20-100 pug)and NPG (1 mg) in a total volume of 1 ml O-01 M sodium acetate buffer, pH 4.2. Reagents were preheated at 6o”C, enzyme was added and the reaction was allowed to proceed for 15 min at 60°C. The reaction was stopped by adding 10 ml of 1% Na,CO, and the absorbance was read at 400 nm.
(ii) beta-Glucosiohse beta-Glucosidase was assayed acccording to Honda et al7 The reaction was initiated by adding 1.9 ml of substrate, (PNGP, 10 111~) dissolved in 50 lll~ sodium acetate buffer, pH 5.0, to O-1 ml of appropriately diluted enzyme (O-5-4 pug) at 50°C. The reaction was carried out for 10 min and terminated by adding 1 ml of 1 M Na,CO,. The yellow colour was read at 400 nm. Chemical modification of enzymes Modification of enzyme with PMDA was carried out according to the procedure described by Mozhaev et aZ.*.
PMDA solution (20 mg ml-‘) in DMSO was added to 1 ml enzyme (amyloglucosidase/betaglucosidase) in O-1 M sodium phosphate buffer, pH 7.0. The pH of the reaction mixture was kept constant by addition of NaOH solution and the reaction was allowed to proceed for 1 h at 25°C. The enzyme solution was then dialyzed against 20 mu sodium acetate buffer, pH 4.2 in the of amyloglucosidase and against 50 ells sodium acetate buffer, pH 5.0 in the case of beta-glucosidase. Immobilization of enzyme on DEAE-cellulose Two millilitres of native/modified enzyme (1 mg/ml) were added to 2 ml settled resin equilibrated with their respective assay buffers and the mixture was stirred for 2 h at 25°C. The resin was then centrifuged at 5000 rpm for 10 min. The ion exchanger containing bound enzyme was washed with the buffer until no enzyme activity could be detected in the washings. Amino group estimation The TNEIS (trinitrobenzene sulphonic acid) method was used for the estimation of free amino group in the protein9.
RESULTS
AND DISCUSSION
Reversibility of immobilization is considered an advantage in many situations such as applications of immobilized enzymes in food industries2. Adsorption on ion exchangers, mostly based upon electrostatic interactions, is a promising strategy for attempting reversible immobilization. However, in order to be a realistic approach, the enzyme should bind to the ion exchangers in an adequate fashion. To facilitate this, chemical modification by acid anhydrides may’ be a useful approach as it enhances negative charges on the enzyme surface and the chemical derivatives are expected to bind to anion exchangers like DEAEcellulose with greater strength. The results obtained with amyloglucosidase and beta- glucosidase using this approach are discussed below.
(i) Amyloglucosidase Modification of amyloglucosidase by PMDA was carried out using different concentrations of PMDA and conditions were optimized to obtain a derivative having an appreciable amount of enzyme activity after adequate modification. The results are shown in Table 1. For further work, the
Immobilization
of chemically
modified
Table 1. Amino group modification of arnyloglucosidase
by
PMDA (see footnote)
Amino group modification (“6)
PMDA (ms)
Residual enzyme activity fW
0.4 0.8
100 100
19
100 100 90
33 33 49
1-2 2.0
Different concentrations of PMDA were added to 1 ml enzyme solution (1 mg/ml) in a O-1 M phosphate buffer, pH 7.0 and incubated for 1 h. The solution was then dialyzed against 20 mM sodium acetate buffer, pH 4,2. The number of free amino groups were then measured by TNBS methodY and the residual enzyme activity was measured using NPC as substrate.”
derivative having 49% amino groups modified with 90% residual enzyme activity was chosen.
amyloglucosidase
445
and beta-glucosidase
Table 2. Elution of native and PMDA modified amyloglucosidase from DEAE-cellulose (see footnote) NaCI concentrations
Enzyme activity eluted fi)
used to eiute the bound protein (M)
Native enzyme bound to DEAE-cellulose
PMDA modified enzyme bound to DEAE-cellulose
36 64
0 0
0.15
0
0
0.2 025 0.5
Z 0
9: 0
0.05 0.1
From 1 ml native/PMDA modified enzyme bound to DEAE-cellulose, the enzyme was eluted by increasing ionic strength of buffer. Different concentrations of NaCl (O-05-0.5 M) were prepared in 20 mM sodium acetate buffer, pH 4.2. The resin was fist incubated in buffer containing @05 M NaCl at 25°C with constant stirring for 30 min. It was then washed with the same buffer until no protein could be detected in the washing. The same resin was then incubated with the buffer containing different concentrations of NaCl (0.1,0.15,0*2,0.25 and @5 M) and was processed in the same way as described above.
Binding to DEAE-cellulose The binding of native and modified enzymes to DEAE-cellulose was checked by adding the same amount of enzyme (1 mg) to a fixed amount of DEAE-cellulose (1 ml settled). About 94% and 99% of the enzyme activity were found to bind DEAE-cellulose in the case of native and PMDA modified enzymes, respectively. The binding in both cases was found to be reversible since the bound enzyme could be eluted out by increasing the ionic strength of the buffer, The elution was done batchwise and the results are shown in Table 2. In the case of native enzyme about 36% enzyme activity was eluted out with O-05 M NaCl and 64% with 0.1 M NaCl. However, the modified enzyme could only be eluted out at higher ionic strength, i.e. with O-25 M NaCl, and thus showing stronger binding to DEAE-cellulose than the native enzyme.
Thermal stability It has been reported that chemical modification of chymotrypsin with PMDA leads to dramatic thermostabilizationB*‘*. This was stated to be the result of hydrophilization of the protein surface as a result of increase in surface charges upon acylation by PMDA. However, in the case of amyloglucosidase the thermostabilization is not significant as the modified enzyme lost about 8 1% activity as compared to the native enzyme which lost 90% of its activity at 70°C in 2 h (Fig.1).
20
0,
-
0
*
’ 30
.
’ 60
Time
(minutes)
.
’ 90
I
1 120
Fig. 1. Thermal stability of native (M) and PMDA modified (H) amyloglucosidase at 70°C. Different enzyme preparations were incubated for 2 h at 70°C at a protein concentration of 1 mg/ml. The residual enzyme activity was measured using NFG as substrate in aliquots withdrawn at different time intervals.
The thermal stability of DEAE-cellulose bound enzyme was checked at 60°C. One millilitre of DEAE-cellulose bound enzyme (native/ PMDA modified) was incubated for 1 h at 60°C and the enzyme activity in the bound form was checked. The PMDA modified enzyme bound to DEAE-cellulose retained full activity while the
Rem
446
Tyagi, M. N. Gupta
bound native enzyme did not show any activity. The loss of enzyme activity in the immobilized preparation could be either due to inactivation as such or due to simple desorption of enzyme from the matrix. To check the latter possibility, after incubation at 60°C for 1 h the resin was washed four times, each with 2 ml of assay buffer. The protein concentration in the supernatant and washings was measured and it was found that 97% of the bound enzyme was leaching out on heating at 60°C for 1 h. Presumably, the enzyme unfolded at 60°C on longer incubation and the unfolded enzyme did not bind to the matrix adequately. The effect of temperature on desorption of enzymes/proteins in the case of noncovalently immobilized enzymes/proteins is not widely studied and such desorption may be a widespread phenomenon. In the case of modified enzyme, Table 3.
Amino
PMDA (m@
1 2 4 6 8
group modification of B-glucosidase PMDA (see footnote)
by
Residual enzyme activity (W
Amino group modification fl)
100 100 97 92 69 62
12 19 30 38 42
Different concentrations of PMDA were added to 1 ml enzyme solution (1 mg/ml in 0.1 M phosphate buffer, pH 8.0). After 1 h incubation at lo”C, the enzyme solution was dialyzed extensively with 50 IIIM sodium acetate buffer, pH 5.0. The number of free amino groups was then measured by TNBS method9 and the residual enzyme activity by using PNGP as substrate.’
stronger binding with the matrix presumably prevented unfolding of the protein and hence the enzyme did not leach out in solution. (ii) beta-Glucosidase The conditions for PMDA modification of beta-glucosidase were also optimized and the results are shown in Table 3. The modified preparation which was used for further studies was the one that showed 92% residual enzyme activity on 30% amino group modification. The idea again is to choose a derivative which retains maximum activity upon sufficient modification. Binding to DEAE-cellulose The native beta-glucosidase does not bind to DEAE-cellulose. However, PMDA modified enzyme showed 95% binding. For optimization of binding of PMDA modified enzyme to DEAEcellulose, varying amounts of modified enzyme were added to the same amount of exchanger and the yield of the immobilized preparation (B/A ) was calculated in terms of expected and obtained activities. The results are shown in Table 4. The best preparation corresponds to a B/A ratio of 40%. The binding of PMDA modified enzyme with DEAE-cellulose was found to be reversible as 97% of the bound protein could be eluted out by increasing the ionic strength of buffer, i.e. at about 0.2 M NaCl (Fig.2). Figure 3 compares the pH activity profiles of native, modified and DEAE-cellulose bound beta-glucosidase. Although native enzyme exhibited a narrow pH zone of maximal activity towards PNGP, the PMDA modified and bound enzymes
Table 4. Binding of PMDA modified B-glucosidase
to DEAE-cellulose
(see footnote)
PMDA modified /?-glucosidase added per 2 ml cellulose (O.D. units) ‘X’
/I-Glucosidase in washings (O.D. units) ‘Y
Bound theoretical (0. D. units) ;y’- ‘Y’=A
Activity of complex (O.D. units) B
B/A x IO0
236 472 706 944
10.28 2026 32.40 60.52
225.72 451.74 673.60 883.48
65.46 171.66 264.44 282.7 1
29 38 40 32
Diierent amounts of enzyme were added to 2 ml settled resin equilibrated with 50 mu sodium acetate buffer, pH 5.0, and incubated for 1 h at 1 O’C. The resin was then washed with equilibrating buffer until no enzyme activity could be detected in the washings. To measure the activity of DEAE-cellulose bound &$ucosidase, the assay mixture was shaken for 10 min at 50°C before the reaction was terminated by 1 M Na,CO,. The O.D. unit here is defined as the product of absorbance obtained by assaying a particular enzyme using an aliquot of 0.1 ml and the total volume of enzyme preparation.
Immobilization
^
0.5
-
0.4
-
of chemically modified amyloglucosidase and beta-glucosidase
447
5 8 -’
0.3 -
0
z
f.
s ‘: 0.2 s
-
- 0.2
-
- 0.1
z 0.1
0
4
0
12 Fraction
16
* m
24
28
number
Fig. 2. Elution profile of PMDA modified beta-glucosidase on DEAE-cellulose column. PMDA modified enzyme (5 mg) was applied on a DEAE-cellulose column equilibrated with 50 mM sodium acetate buffer, pH 5.0. The arrow indicates start of NaCl gradient (O-O.5 M in sodium acetate buffer). Fractions of 3 ml were collected at a flow rate of 20 ml/h.
0.5
-
0
10
20
30
CO
50
PH
Time (minutes)
Fig. 3. Effect of pH on the activity of native (U), PMDA modified (c---l ) and DEAE-cellulose bound (A-A ) beta-glucosidase. The activity of beta-glucosidase was measured in 50 mu citrate buffer for pH 3.0, 50 mM sodium acetate buffer for pH 4*0-6-O and 50 ~IIMphosphate buffer for pH 7.0. and 8.0.
Fig. 4. Thermal stability of native (M), PMDA modified (u) and DEAE-cellulose bound (A-A ) beta-glucosidase. Different enzyme preparations were incubated at 65°C for 50 mm at a protein concentration of 0.1 mg/ml. The residual enzyme activity was measured using PNGP as substrate in aliquots withdrawn at different time intervals.
had slightly broader pH activity profiles with the pH optimum shifted slightly towards the acidic side. Thermal stability The thermal stability of different preparations of beta-glucosidase were studied in 50 mu sodium acetate buffer, pH 5.0 at 65°C for 50 min. The PMDA modified and bound enzymes showed
enhancement in thermal stability over the native enzyme (Fig.4). In the case of an immobilized system, the enhanced thermostability is not of great value in view of reduction in enzyme activity as a result of immobilization. However, the soluble modified enzyme does show the effect of surface hydrophihzation on thermal stability as predicted by the Mozhaev et aI.8.
448
Renu Tyagi, M. N. Gupta
Thus, hydrophilization by PMDA does not always lead to thermostabihzation. Earlier, the authors reported that trypsin did not show much of a thermostabilization upon modification with PMDA. In the present work, the data show that amyloglucosidase also did not show any significant increase in thermal stability. In the case of however, significant improvebeta-glucosidase, ment in thermal stability was observed. On the other hand, in the three enzymes studied so far, reasonable level of chemical modification (with very little loss in activity) by PMDA yielded enzyme derivatives which showed improved binding to DEAE-cellulose. Also, PMDA does appear to be a better reagent as compared to succinic anhydride since in the work of Solomon and Levin4 succinylated enzyme retained only 60% activity. With the same enzyme, in the authors’ case acylation with PMDA led to an enzyme derivative which retained 90% activity. Thus, acylation by PMDA may be a useful general strategy for binding enzymes to anion exchangers.
ACKNOWLEDGEMENTS
REFERENCES 1. Rosevear,
biocatalysis. In Molecular ed. J. M. Walker & E. B. Gingold. Royal Sot. Chem., London, 1988, pp. 243-5. 2. Gupta, M. N. & Mattiasson, B., Unique applications of immobilized proteins in bioanalytical systems. In Bioanalytical Application of Enzymes. Vol.36, ed. C. H. Suelter. John Wiley, New York, 1992, pp. l-36. 3. Carbal, J. M. S. & Kennedy, J. F., Immobilization techniques for altering thermal stability of enzymes. In Thermostability of Enzymes. ed. M. N. Gupta. Springer Verlag, Germany, pp. 162-9. 4. Solomon, B. & Levin, Y., Studies on adsorption of amyloglucosidase on ion-exchange resin. Biotechnol.
Immobilized
Bioeng., 16 (1974) 1161-77. 5. Goldstein, L. & Manecke, G., The chemistry of enzyme immobilization. In Applied biochemistry and bioengineering. Vol.1, ed. L. B. Wingard, E. KatchalskiKatzir & L. Goldstein. Academic Press, New York, 1976, pp. 23-126. 6. Stemberg, H. Z. The separation of proteins with heteropolyacids. Biotechnol. Bioeng., 12 (1970) l-17. 7. Honda, H., Saito, T., Iijima, S. & Kobayashi, T., Molecular cloning and expression of a beta-glucosidase gene from Ruminococcus albuus in E. Coli. Enzyme Microb.
Technol., 10 (1988) 559-62. 8. Mozhaev, V. V., Siksnis, V. A., Melik-Nubarov,
N. S., Galkantaite, N. Z., Denis, G. J., Butkus, E. P., Zaslavsky, B. V. Mestectikina, N. M. & Martinek, K., Protein stabilization via hydrophilization. Eur. J. Biochem., 173
(1988) 147-54. 9. Habeeb, A. F. S. A., Determination in
This work was supported by the Council of Scientific and Industrial Research (CSIR), India, in the form of a research grant to one of the authors (MNG).
A.,
Biology and Biotechnology
proteins
by
of free ammo groups trinitrobenzenesulfonic acid, Anal.
Biochem., 14 (1966) 328-36. N. S. Levitsky V. Y., 10. Mozhaev, V. V., Melik-Nubarov, Siksnis, V. A. and Martinek, K., High stability to irreversible inactivation at elevated temperatures of enzymes covalently modified by hydrophilic reagents: achymotrypsin. Biotechnol. Bioeng., 40 (1992) 650-62.
Printedin Great Britain.All tightsreserved O32-9592/94/$7.00
Noncovalent and Reversible Immobilization of Chemically Modified Amyloglucosidase and betaGlucosidase on DEAE-Cellulose Renu Tyagi & M. N. Gupta* Chemistry Department, (Received
Indian Institute of Technology,
14 June 1993; accepted
Delhi, New Delhi-1 10 016, India
8 July 1993)
The acylation of amino groups of proteins by pyromellitic dianhydtide (PMDA) leads to an increase in negative charges on the protein surface by 4 units for every amino group modified. This reaction has been used to mod@ enzymes for the pqvose of adsorption on DEAE-cellulose. The 49% amino groups of amyloglucoskiase were modijied with 90% residual enzyme activity. Whereas the native enzyme bound to DEAE-cellulose and could be eluted out with 0.1 M sodium chloride, the modeed enzyme exhibited stronger binding and could only be eluted with 0.25’ M sodium chloride. In the case of a&orbed native enzyme, heating at 60°C for 1 h led to desorption of 97% protein. In the case of the modified enzyme, the immobilized preparation retainedfull enzyme activity under similar conditions. beta-Glucosidase (which did not bind to DEAE-cellulose) upon modification with PMDA was found to bind to DEAE-cellulose and could be eluted out with 97% protein recovery by washing with O-2 M sodium chloride. Thus, chemical modification with PMDA may be a useful and general strategy for obtaining enzyme derivatives for reversible adriorption on anion exchangers.
INTRODUCTION
sufficient. Chemical modification of the protein to create appropriate charges on the surface is an obvious strategy which has been occasionally employed4J. Solomon and Levin4 used succinic anhydride to modify amyloglucosidase and the modified amyloglucosidase was bound to DEAEcellulose. Recently, the present authors have modified trypsin with both succinic anhydride and pyromellitic dianhydride (PMDA); the modified enzyme (Scheme 1), unlike the native one, bound to DEAE-cellulose. Pyromellitic dianhydride was found to be a better reagent for this purpose since for the same extent of chemical modification, the enzyme
Adsorption on ion exchangers constitutes a fairly successful approach to enzyme immobilization for industrial application’. The advantages of such noncovalent methods for protein/enzyme immobilization have been reviewed recently2+3. It is not always feasible to bind an enzyme on an ion exchanger with adequate strength as the surface charges on the enzyme may not be appropriate or
*Corresponding author: M. N. Gupta. Telephone: 9 1 11 666 979 ext.7615. Fax: 91 11686 2037.
443
444
R-NH2
Renu Tyagi, M. N. Gupta
Protein
RNH-CO
coo-
-0oc
coo-
o-
+
PMOA moditied deriwtii
PMOA
Scheme
1.
modified with PMDA retained significantly higher activity than the enzyme modified with succinic anhydride. In this work, the authors extend their approach to immobilize two biotechnologically useful enzymes ,viz. amyloglucosidase and betaglucosidase on DEAE-cellulose.
MATERIAL
AND METHODS
Amyloglucosidase (from Aspergillus n&r), beta- glucosidase, (from almonds), p-nitrophenyl alpha-n-glucopyranoside (NPG) and p-nitrophenyl beta-n-glucopyranoside (PNGP) were purchased from Sigma Chemical Co., USA. Pyromellitic dianhydride (PMDA) was a product of Eastman Kodak Co., USA (the authors thank Prof. A. M. Klibanov, MIT, USA for the gift of this chemical). All other reagents were of analytical grade. Enzyme Assay (i) Amyloglucosidase The enzyme assay was performed using NPG as substrate”. Each assay contained enzyme (20-100 pug)and NPG (1 mg) in a total volume of 1 ml O-01 M sodium acetate buffer, pH 4.2. Reagents were preheated at 6o”C, enzyme was added and the reaction was allowed to proceed for 15 min at 60°C. The reaction was stopped by adding 10 ml of 1% Na,CO, and the absorbance was read at 400 nm.
(ii) beta-Glucosiohse beta-Glucosidase was assayed acccording to Honda et al7 The reaction was initiated by adding 1.9 ml of substrate, (PNGP, 10 111~) dissolved in 50 lll~ sodium acetate buffer, pH 5.0, to O-1 ml of appropriately diluted enzyme (O-5-4 pug) at 50°C. The reaction was carried out for 10 min and terminated by adding 1 ml of 1 M Na,CO,. The yellow colour was read at 400 nm. Chemical modification of enzymes Modification of enzyme with PMDA was carried out according to the procedure described by Mozhaev et aZ.*.
PMDA solution (20 mg ml-‘) in DMSO was added to 1 ml enzyme (amyloglucosidase/betaglucosidase) in O-1 M sodium phosphate buffer, pH 7.0. The pH of the reaction mixture was kept constant by addition of NaOH solution and the reaction was allowed to proceed for 1 h at 25°C. The enzyme solution was then dialyzed against 20 mu sodium acetate buffer, pH 4.2 in the of amyloglucosidase and against 50 ells sodium acetate buffer, pH 5.0 in the case of beta-glucosidase. Immobilization of enzyme on DEAE-cellulose Two millilitres of native/modified enzyme (1 mg/ml) were added to 2 ml settled resin equilibrated with their respective assay buffers and the mixture was stirred for 2 h at 25°C. The resin was then centrifuged at 5000 rpm for 10 min. The ion exchanger containing bound enzyme was washed with the buffer until no enzyme activity could be detected in the washings. Amino group estimation The TNEIS (trinitrobenzene sulphonic acid) method was used for the estimation of free amino group in the protein9.
RESULTS
AND DISCUSSION
Reversibility of immobilization is considered an advantage in many situations such as applications of immobilized enzymes in food industries2. Adsorption on ion exchangers, mostly based upon electrostatic interactions, is a promising strategy for attempting reversible immobilization. However, in order to be a realistic approach, the enzyme should bind to the ion exchangers in an adequate fashion. To facilitate this, chemical modification by acid anhydrides may’ be a useful approach as it enhances negative charges on the enzyme surface and the chemical derivatives are expected to bind to anion exchangers like DEAEcellulose with greater strength. The results obtained with amyloglucosidase and beta- glucosidase using this approach are discussed below.
(i) Amyloglucosidase Modification of amyloglucosidase by PMDA was carried out using different concentrations of PMDA and conditions were optimized to obtain a derivative having an appreciable amount of enzyme activity after adequate modification. The results are shown in Table 1. For further work, the
Immobilization
of chemically
modified
Table 1. Amino group modification of arnyloglucosidase
by
PMDA (see footnote)
Amino group modification (“6)
PMDA (ms)
Residual enzyme activity fW
0.4 0.8
100 100
19
100 100 90
33 33 49
1-2 2.0
Different concentrations of PMDA were added to 1 ml enzyme solution (1 mg/ml) in a O-1 M phosphate buffer, pH 7.0 and incubated for 1 h. The solution was then dialyzed against 20 mM sodium acetate buffer, pH 4,2. The number of free amino groups were then measured by TNBS methodY and the residual enzyme activity was measured using NPC as substrate.”
derivative having 49% amino groups modified with 90% residual enzyme activity was chosen.
amyloglucosidase
445
and beta-glucosidase
Table 2. Elution of native and PMDA modified amyloglucosidase from DEAE-cellulose (see footnote) NaCI concentrations
Enzyme activity eluted fi)
used to eiute the bound protein (M)
Native enzyme bound to DEAE-cellulose
PMDA modified enzyme bound to DEAE-cellulose
36 64
0 0
0.15
0
0
0.2 025 0.5
Z 0
9: 0
0.05 0.1
From 1 ml native/PMDA modified enzyme bound to DEAE-cellulose, the enzyme was eluted by increasing ionic strength of buffer. Different concentrations of NaCl (O-05-0.5 M) were prepared in 20 mM sodium acetate buffer, pH 4.2. The resin was fist incubated in buffer containing @05 M NaCl at 25°C with constant stirring for 30 min. It was then washed with the same buffer until no protein could be detected in the washing. The same resin was then incubated with the buffer containing different concentrations of NaCl (0.1,0.15,0*2,0.25 and @5 M) and was processed in the same way as described above.
Binding to DEAE-cellulose The binding of native and modified enzymes to DEAE-cellulose was checked by adding the same amount of enzyme (1 mg) to a fixed amount of DEAE-cellulose (1 ml settled). About 94% and 99% of the enzyme activity were found to bind DEAE-cellulose in the case of native and PMDA modified enzymes, respectively. The binding in both cases was found to be reversible since the bound enzyme could be eluted out by increasing the ionic strength of the buffer, The elution was done batchwise and the results are shown in Table 2. In the case of native enzyme about 36% enzyme activity was eluted out with O-05 M NaCl and 64% with 0.1 M NaCl. However, the modified enzyme could only be eluted out at higher ionic strength, i.e. with O-25 M NaCl, and thus showing stronger binding to DEAE-cellulose than the native enzyme.
Thermal stability It has been reported that chemical modification of chymotrypsin with PMDA leads to dramatic thermostabilizationB*‘*. This was stated to be the result of hydrophilization of the protein surface as a result of increase in surface charges upon acylation by PMDA. However, in the case of amyloglucosidase the thermostabilization is not significant as the modified enzyme lost about 8 1% activity as compared to the native enzyme which lost 90% of its activity at 70°C in 2 h (Fig.1).
20
0,
-
0
*
’ 30
.
’ 60
Time
(minutes)
.
’ 90
I
1 120
Fig. 1. Thermal stability of native (M) and PMDA modified (H) amyloglucosidase at 70°C. Different enzyme preparations were incubated for 2 h at 70°C at a protein concentration of 1 mg/ml. The residual enzyme activity was measured using NFG as substrate in aliquots withdrawn at different time intervals.
The thermal stability of DEAE-cellulose bound enzyme was checked at 60°C. One millilitre of DEAE-cellulose bound enzyme (native/ PMDA modified) was incubated for 1 h at 60°C and the enzyme activity in the bound form was checked. The PMDA modified enzyme bound to DEAE-cellulose retained full activity while the
Rem
446
Tyagi, M. N. Gupta
bound native enzyme did not show any activity. The loss of enzyme activity in the immobilized preparation could be either due to inactivation as such or due to simple desorption of enzyme from the matrix. To check the latter possibility, after incubation at 60°C for 1 h the resin was washed four times, each with 2 ml of assay buffer. The protein concentration in the supernatant and washings was measured and it was found that 97% of the bound enzyme was leaching out on heating at 60°C for 1 h. Presumably, the enzyme unfolded at 60°C on longer incubation and the unfolded enzyme did not bind to the matrix adequately. The effect of temperature on desorption of enzymes/proteins in the case of noncovalently immobilized enzymes/proteins is not widely studied and such desorption may be a widespread phenomenon. In the case of modified enzyme, Table 3.
Amino
PMDA (m@
1 2 4 6 8
group modification of B-glucosidase PMDA (see footnote)
by
Residual enzyme activity (W
Amino group modification fl)
100 100 97 92 69 62
12 19 30 38 42
Different concentrations of PMDA were added to 1 ml enzyme solution (1 mg/ml in 0.1 M phosphate buffer, pH 8.0). After 1 h incubation at lo”C, the enzyme solution was dialyzed extensively with 50 IIIM sodium acetate buffer, pH 5.0. The number of free amino groups was then measured by TNBS method9 and the residual enzyme activity by using PNGP as substrate.’
stronger binding with the matrix presumably prevented unfolding of the protein and hence the enzyme did not leach out in solution. (ii) beta-Glucosidase The conditions for PMDA modification of beta-glucosidase were also optimized and the results are shown in Table 3. The modified preparation which was used for further studies was the one that showed 92% residual enzyme activity on 30% amino group modification. The idea again is to choose a derivative which retains maximum activity upon sufficient modification. Binding to DEAE-cellulose The native beta-glucosidase does not bind to DEAE-cellulose. However, PMDA modified enzyme showed 95% binding. For optimization of binding of PMDA modified enzyme to DEAEcellulose, varying amounts of modified enzyme were added to the same amount of exchanger and the yield of the immobilized preparation (B/A ) was calculated in terms of expected and obtained activities. The results are shown in Table 4. The best preparation corresponds to a B/A ratio of 40%. The binding of PMDA modified enzyme with DEAE-cellulose was found to be reversible as 97% of the bound protein could be eluted out by increasing the ionic strength of buffer, i.e. at about 0.2 M NaCl (Fig.2). Figure 3 compares the pH activity profiles of native, modified and DEAE-cellulose bound beta-glucosidase. Although native enzyme exhibited a narrow pH zone of maximal activity towards PNGP, the PMDA modified and bound enzymes
Table 4. Binding of PMDA modified B-glucosidase
to DEAE-cellulose
(see footnote)
PMDA modified /?-glucosidase added per 2 ml cellulose (O.D. units) ‘X’
/I-Glucosidase in washings (O.D. units) ‘Y
Bound theoretical (0. D. units) ;y’- ‘Y’=A
Activity of complex (O.D. units) B
B/A x IO0
236 472 706 944
10.28 2026 32.40 60.52
225.72 451.74 673.60 883.48
65.46 171.66 264.44 282.7 1
29 38 40 32
Diierent amounts of enzyme were added to 2 ml settled resin equilibrated with 50 mu sodium acetate buffer, pH 5.0, and incubated for 1 h at 1 O’C. The resin was then washed with equilibrating buffer until no enzyme activity could be detected in the washings. To measure the activity of DEAE-cellulose bound &$ucosidase, the assay mixture was shaken for 10 min at 50°C before the reaction was terminated by 1 M Na,CO,. The O.D. unit here is defined as the product of absorbance obtained by assaying a particular enzyme using an aliquot of 0.1 ml and the total volume of enzyme preparation.
Immobilization
^
0.5
-
0.4
-
of chemically modified amyloglucosidase and beta-glucosidase
447
5 8 -’
0.3 -
0
z
f.
s ‘: 0.2 s
-
- 0.2
-
- 0.1
z 0.1
0
4
0
12 Fraction
16
* m
24
28
number
Fig. 2. Elution profile of PMDA modified beta-glucosidase on DEAE-cellulose column. PMDA modified enzyme (5 mg) was applied on a DEAE-cellulose column equilibrated with 50 mM sodium acetate buffer, pH 5.0. The arrow indicates start of NaCl gradient (O-O.5 M in sodium acetate buffer). Fractions of 3 ml were collected at a flow rate of 20 ml/h.
0.5
-
0
10
20
30
CO
50
PH
Time (minutes)
Fig. 3. Effect of pH on the activity of native (U), PMDA modified (c---l ) and DEAE-cellulose bound (A-A ) beta-glucosidase. The activity of beta-glucosidase was measured in 50 mu citrate buffer for pH 3.0, 50 mM sodium acetate buffer for pH 4*0-6-O and 50 ~IIMphosphate buffer for pH 7.0. and 8.0.
Fig. 4. Thermal stability of native (M), PMDA modified (u) and DEAE-cellulose bound (A-A ) beta-glucosidase. Different enzyme preparations were incubated at 65°C for 50 mm at a protein concentration of 0.1 mg/ml. The residual enzyme activity was measured using PNGP as substrate in aliquots withdrawn at different time intervals.
had slightly broader pH activity profiles with the pH optimum shifted slightly towards the acidic side. Thermal stability The thermal stability of different preparations of beta-glucosidase were studied in 50 mu sodium acetate buffer, pH 5.0 at 65°C for 50 min. The PMDA modified and bound enzymes showed
enhancement in thermal stability over the native enzyme (Fig.4). In the case of an immobilized system, the enhanced thermostability is not of great value in view of reduction in enzyme activity as a result of immobilization. However, the soluble modified enzyme does show the effect of surface hydrophihzation on thermal stability as predicted by the Mozhaev et aI.8.
448
Renu Tyagi, M. N. Gupta
Thus, hydrophilization by PMDA does not always lead to thermostabihzation. Earlier, the authors reported that trypsin did not show much of a thermostabilization upon modification with PMDA. In the present work, the data show that amyloglucosidase also did not show any significant increase in thermal stability. In the case of however, significant improvebeta-glucosidase, ment in thermal stability was observed. On the other hand, in the three enzymes studied so far, reasonable level of chemical modification (with very little loss in activity) by PMDA yielded enzyme derivatives which showed improved binding to DEAE-cellulose. Also, PMDA does appear to be a better reagent as compared to succinic anhydride since in the work of Solomon and Levin4 succinylated enzyme retained only 60% activity. With the same enzyme, in the authors’ case acylation with PMDA led to an enzyme derivative which retained 90% activity. Thus, acylation by PMDA may be a useful general strategy for binding enzymes to anion exchangers.
ACKNOWLEDGEMENTS
REFERENCES 1. Rosevear,
biocatalysis. In Molecular ed. J. M. Walker & E. B. Gingold. Royal Sot. Chem., London, 1988, pp. 243-5. 2. Gupta, M. N. & Mattiasson, B., Unique applications of immobilized proteins in bioanalytical systems. In Bioanalytical Application of Enzymes. Vol.36, ed. C. H. Suelter. John Wiley, New York, 1992, pp. l-36. 3. Carbal, J. M. S. & Kennedy, J. F., Immobilization techniques for altering thermal stability of enzymes. In Thermostability of Enzymes. ed. M. N. Gupta. Springer Verlag, Germany, pp. 162-9. 4. Solomon, B. & Levin, Y., Studies on adsorption of amyloglucosidase on ion-exchange resin. Biotechnol.
Immobilized
Bioeng., 16 (1974) 1161-77. 5. Goldstein, L. & Manecke, G., The chemistry of enzyme immobilization. In Applied biochemistry and bioengineering. Vol.1, ed. L. B. Wingard, E. KatchalskiKatzir & L. Goldstein. Academic Press, New York, 1976, pp. 23-126. 6. Stemberg, H. Z. The separation of proteins with heteropolyacids. Biotechnol. Bioeng., 12 (1970) l-17. 7. Honda, H., Saito, T., Iijima, S. & Kobayashi, T., Molecular cloning and expression of a beta-glucosidase gene from Ruminococcus albuus in E. Coli. Enzyme Microb.
Technol., 10 (1988) 559-62. 8. Mozhaev, V. V., Siksnis, V. A., Melik-Nubarov,
N. S., Galkantaite, N. Z., Denis, G. J., Butkus, E. P., Zaslavsky, B. V. Mestectikina, N. M. & Martinek, K., Protein stabilization via hydrophilization. Eur. J. Biochem., 173
(1988) 147-54. 9. Habeeb, A. F. S. A., Determination in
This work was supported by the Council of Scientific and Industrial Research (CSIR), India, in the form of a research grant to one of the authors (MNG).
A.,
Biology and Biotechnology
proteins
by
of free ammo groups trinitrobenzenesulfonic acid, Anal.
Biochem., 14 (1966) 328-36. N. S. Levitsky V. Y., 10. Mozhaev, V. V., Melik-Nubarov, Siksnis, V. A. and Martinek, K., High stability to irreversible inactivation at elevated temperatures of enzymes covalently modified by hydrophilic reagents: achymotrypsin. Biotechnol. Bioeng., 40 (1992) 650-62.