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Characterization of a glucoamylase immobilized on chitin
Biomass 23 (1990) 71-78
Short Communication Characterization of a Giucoamylase Immobilized on Chitin
ABSTRACT Glucoamylase (E.C. 3.2.1.3) covalently immobilized on chitin panicles (24-60 mesh) showed an average activity of lO00 U g- 1. Temperature and pH optima were 60°C and 3"6, respectively. These values were lower than the corresponding ones for the free enzyme (pH 4"4, temperature 65°C). The K m (Michaelis constant) values for soluble starch and hydrolyzed manioc starch were, respectively, 1"25 and 3"94 g litre- i (free enzyme) and 8"6 and 7.8g litre i (immobilized enzyme). Key words: glucoamylase, enzyme immobilization, manioc starch hydrolysis.
INTRODUCTION The production of glucose from starch by enzymatic processes is growing in commercial importance. The use of immobilized glucoamylase in the saccharification step of these processes has some advantages: it enables continuous operation with reduced manpower and reduces enzyme use. However, the use of immobilized enzymes in industrial processes depends on the availability of an active and stable biocatalyst. This note reports the evaluation and characterization of a biocatalyst whose preparation, described in a previous study by Bon et al.,~ was subsequently improved. The literature contains numerous reports on the use of many types of support materials and methods of immobilization for glucoamylase. Table 1 summarizes some of the more recently published works. Chitin, the support material used in this study, has previously been used for immobilization of glucoamylase by Stanley et al. 2 and by Flor and Hayashida. 3 71 Biomass 0144-4565/90/S03.50 © 1990 Elsevier Science Publishers Ltd, England. Printed in Great Britain
72
D. G. Freire, G. L. Sant'Anna Jr
MATERIALS A N D METHODS
Glucoamylase, from Aspergillus niger (AMG Novo L-Novo Industri, Bagsvaerd, Denmark), was immobilized on chitin particles (mean diameter 0.37 mm) activated with hexamethylenediamine and glutaraldehyde following the procedure described by Bon et aL 1 Two types of substrates were used: soluble starch (analytical grade: Reagen) and commercial manioc starch flour, which was enzymatically hydrolyzed with a-amylase (E.C. 3.2.1.1)(Termamyl-Novo Industri, Bagsvaerd, Denmark) using a ratio of 0.1 w/w enzyme/starch. The experimental set-up used for the evaluation of the biocatalyst was as described in a previous paper) 1 The reactor was a small stirred vessel (60 ml) with a removable stainless steel basket (150 mesh) submerged in the liquid. Samples of approximately 40 mg of the biocatalyst particles were put inside the basket and its activity measured as the initial rate of glucose production. One unit of activity was defined as the amount of enzyme which produce 1 pmol of glucose min-~, at 45°C, in buffered (Na acetate 0.1 M) solutions of substrate. Glucose was determined by the glucoseoxydase/peroxydase test (GOD-PAP Merckotest 3395,
TABLE 1 Some Published Works on Glucoamylase Immobilization Support
Half-life
Substrate
Reference
time
Polyethylene vinyl alcohol D E A E - - cellulose
> 150 days (5°C) --
Porous glass fiber Polymethacrylate
8 days --
Corn stover
40 days 105 days (60°C)
Polyvinyl
Imai et al. 4
Soluble starch Maltose
Tomar & Prabhu 5 Toldra et al. 6
(50°C)
(400C)
Polyacrylamide
Maltose
2.5 h (40°C) 60 days (4°C)
Soluble starch Hydrolyzed starch Soluble and hydrolyzed starch Maltose starch
Fiedureck et al. 7 Vallat & Monsan 8 Szajani et aL 9
Imai et aL 10
Character&ation of a glucoamylase immobilized on chitin
73
Darmstadt, Austria). The parameters associated with glucoamylase in solution (free enzyme) were determined using the same methods.
RESULTS AND DISCUSSION The influence of pH on enzyme activity (expressed as relative initial rate of glucose production) is shown in Fig. 1. The immobilized enzyme had an optimum pH value which was lower than that of the free enzyme. This decrease in pH may be related to the existence of positive charges on the surface of the particle, probably associated with residual amino groups. Depending on the ionic strength of the solution, these charges contribute to the establishment of a pH gradient near the particle and thus the immobilized enzyme is submitted to a pH which is different from the pH of the bulk solution. Similar results were observed by Stanley et aL 2 when using chitin activated with glutaraldehyde and by CabraP 2 when using porous silica activated with amine and glutaraldehyde. The influence of temperature on the activity of glucoamylase (free and immobilized) are shown in Fig. 2. The optimum temperature for the immobilized enyzme was 60°C, somewhat lower than the optimum temperature for the free enzyme (65°C). A more rigid molecular structure typical of immobilized enzymes may enhance its sensitivity to temperature inactivation, thus leading to a lower optimum temperature value in comparison with the free enzyme. Other similar results showing differences in optima temperatures for free and immobilized enzymes are reported by Bachler et aL, 13 Maeda and Suzuki, 14 and Szajani et aL 9 The experimental data confined to the ascendant part of the curves shown in Fig. 2 may be represented by an Arrhenius relationship, leading to activation energy values of 12.6 and 6.7 kcal mol -~ for free and relative rote of reaction (%1 100 • @
immobilized enzyme
0 free enzyme
50'
L/ Fig. 1.
Influence of pH on glucoamylase activity.
74
D. G. Freire, G. L. Sant'Anna Jr
immobilized enzymes respectively. A decrease in the activation energy for immobilized enzymes was also reported by Cabral ]2 and Weetal, ]5 who attribute this result to diffusional limitations and conformational effects associated with immobilized biocatalysts. The kinetic parameters of the glucoamylase were determined at 45°C (at the optimal temperature the rate of enzyme inactivation is too high) and optimal pH (4.4 for the free enzyme and 3"6 for the immobilized enzyme). The substrates used were soluble starch and hydrolyzed manioc starch. The experimental data were successfully represented by the classical Michaelis-Menten model. The Lineweaver-Burk plot of these data resulted in straight lines as shown in Figs 3 and 4, with correlation coefficients higher than 0.94. The kinetic parameters obtained from the slope and the intercept of these straight lines are listed in Table 2. From these results, it can be seen that the immobilized enzyme presents higher Km values than the free enzyme. This is related to the increased diffusional resistance and steric effects associated with the immobilized biocatalysts. This same trend was observed by several authors: Park,~6 Nithianandham et al., 17 Tomar and Prabhu, 5 Vallat and Monsan, 8 and Imai et al. 4 The K m value obtained for the free enzyme and soluble starch is close to the values obtained by Kennedy and Epton ~8 and Cabral; ~2 the K m value for the immobilized glucoamylase (8"6 g litre -~ ) is comparable to those reported by CabraP 2 for different supports: celite (5,5 g litre-~), controlled pore glass (8-0 g litre -l) and pumice-stone (6.4 g litre-~). The free enzyme K m value was lower for soluble starch substrate, whose mean molecular weight (MW) is greater than that of the hydrolyzed manioc starch substrate. The decrease of Km with the increase of the substrate MW was also remarked on by Kusunoki et al. ~9and Imai et al. ~o The effect of substrate MW o n K m for immobilized enzymes seems to be less evident and in this experiment a
relative rate of reaction (%) 100
• immobilized enzyme 0 free enzyme
50-
-'// Fig. 2.
:30 40 50 60
~;0 eO
I~emperoture(°c)
Influence of temperature on glucoamylase activity.
Characterization of a glucoamylase immobilized on chitin
75
slight decrease of K m w a s registered with the decrease of substrate MW. This same trend was observed by Vallat and Monsan 8 in experiments with immobilized glucoamylase and two different MW substrates. The VmaX (maximum velocity, Michaelis model) values reported in Table 2
! ~6.0 -- IT
o-FREEENZYME
I- 5.0
~'~
1.0
0.5
i.o
i.5
i
i
i
!
Fig. 3.
.
.
.
.
1/S
(9/IP)-i
z.o i
.
Lineweaver-Burk plot of free and immobilized glucoamylase on soluble starch. S = substrate concentration (g/litre).
TABLE 2 Kinetic Parameters of Immobilized and Free Glucoamylase Enzyme
Free Free
Immobilized Immobilized
Substrate
Soluble starch Hydrolyzed manioc starch Soluble starch Hydrolyzed manioc starch
Km (g litre-/)
Vma× (# mol m i n ¢ g- / support)
Illll ),,
(p mol min- i mg-litre protein)
1.25
56'1
3-94
45"7
8.61
941
7.82
920
76
D. G. Freire, G. L. Sant'Anna Jr
-IT/
~*1
-,..o.,L,zEo
20
.----.--""0""
~........~-
o
" " "
o.1 I
o.2 I
o,~
Fig. 4.
o.a I
l/S (~11)"I r
0.4 |
t/S 10/11 "I
o.1
Lineweaver-Burk plot of free and immobilized glucoamylase on hydrolyzed manioc starch. S = substrate concentration (g/litre).
seem to be little influenced by the substrate MW for the free and immobilized glucoamylase. The biocatalyst used in this work was further tested by Freire 2° under continuous conditions in a laboratory-scale expanded-bed reactor fed with a solution of hydrolyzed manioc starch (15% w/w) at 45°C. A very smooth decrease of the immobilized glucoamylase activity was observed and the experimental data lead to a half-life time of 156 days, a result which is in the range of the best stability data reported in the literature. ACKNOWLEDGEMENT This work was supported by grants of Conselho Nacional de Desenvolvimento Cientffico e Technol6gico, CNPq (Brazil). This support is gratefully acknowledged by the authors. REFERENCES 1. Bon, E., Freire, D. B., Mendes, M. F., Moreira, C. P. & Soares, V. E, Immobilization of glucoamylase on inexpensive supports: A comparative evaluation. Biotechnol. Bioeng., Symposiumno. 14 (1984) 485-92.
Characterization of a glucoamylase immobilized on chitin
77
2. Stanley, W. L., Watters, G. G., Kelly, S. H. & Olson, A. C., Glutaraldehyde immobilized on chitin with glutaraldehyde. Biotechnol. Bioeng., 2t) (1978) 135-40. 3. Flor, P. Q. & Hayashida, S., Production and characteristics of raw-starchdigesting glucoamylase O from a protease -- negative glucosidase negative Aspergillus awanori vat. kaawachi mutant. Applied and Environmental Microb., 45 (3)(1983) 905-12. 4. Imai, K., Shiomi, T., Uchida, K. & Miya, M., Immobilization of enzyme onto polyethylene-vinyl alcohol membrane. Biotechnol. Bioeng., 28 (1986) 198-203. 5. Tomar, M. & Prabhu, K. A., Immobilization of glucoamylase on DEAEcellulose activated with chloride compounds. Enzyme Microb. Technol., 7 (1985) 557-9. 6. Toldra, E, Jansen, N. B. & Tsao, G. T. Use of a porous glass fiber as a support for biocatalyst immobilization. Biotechnol. Letters, $ ( 11 ) (1986) 785-90. 7. Fiedureck, J., Lobarzewski, J., Wojcik, A. & Wolski, T., Optimization of enzyme immobilization on keratin or polyamide-coated bed-shaped polymeric matrix. Biotechnol. Bioeng., 28 (1986) 744-50. 8. Vallat, I. & Monsan, P., Maltodextrin hydrolysis in the fluidized-bed immobilized enzyme reactor. Biotechnol. Bioeng., 23 (1986) 151-9. 9. Szajani, B., Klamar, G. & Ludwig, L., Preparation, characterization and laboratory-scale application of an immobilized glucoamylase. Enzyme Microb. Technol., 7 (1985) 488-92. 10. Imai, K., Shiomi, T., Uchida, K. & Miya, M., Immobilization of enzyme into poly-vinyl-alcohol membrane. Biotechnol. Bioeng., 28 (1986) 1721-6. 11. Xavier, M. T., Soares, V. E, Freire, D. M. G., Moreira, C. P., Mendes, M. E & Bon, E., a-Amylase and glucoamylase immobilized on chitin and ceramic supports. Biomass, 13 (1987) 25-32. 12. Cabral, J. M. S., Estudo de imobilizaqho de enzimas pelo metodo dos metais de transi~o. DSc. thesis, Universidade T6cnica de Lisboa, 1982. 13. Bachler, M. J., Strandberg, G. U. & Similey, K. L., Starch conversion by immobilized glucoamylase. Biotechnol. Bioeng., 12 ( 1970) 85-92. 14. Maeda, J. & Suzuki, H., Preparative of immobilized enzymes by N-vinylpyrrolidone and the general properties of the glucoamylase gel. Biotechnol. Bioeng., 16 (1974) 1517-28. 15. Weetal, H. H., Immobilized enzymes and their application in the food and beverage industry. Process Biochemistry, $ ( 1975 ) 3-30. 16. Park, Y. K., Enzymic properties of fungal amyloglicosidase-resin complex. J. Ferm. Technol., 2 (2)(1974) 140-2. 17. Nithianandham, V. S., Srinivasan, K. S. V., Joseph, K. T. & Santappa, M., Preparation and properties of immobilized amyloglicosidase. Biotechnol. Bioeng., 23 (1982)2273-82. 18. Kennedy, J. F. & Epton, J., Poly N-acryloyl-4 and 5-aminosalicylic acids. Carbohydrate Research, 27 (1973) 11-20. 19. Kusunoki, K., Kawakami, K., Shiraishi, K. & Kai, M., A kinetic expression for hydrolysis of soluble starch by glucoamylase. Biotechnol. Bioeng., 24 (1982) 347-54.
78
D. G. Freire, G. L. Sant'Anna Jr
20. Freire, D. M. G., Imobilizaq~o de amiloglicosidase em quitina. MSc thesis, Escola de Quimica, Universidade Federal do Rio de Janeiro, 1988.
Denise Guimar~es Freire & Geraido Lippel Sant'Anna Jr* COPPE, Escola de Quimica and Instituto de Quimica, Universidade Federal do Rio de Janeiro, PO Box 68502, 21945 Rio de Jane&o, Brazil (Received 19 April 1989; accepted 26 June 1989) *To whom correspondence should be addressed.
Short Communication Characterization of a Giucoamylase Immobilized on Chitin
ABSTRACT Glucoamylase (E.C. 3.2.1.3) covalently immobilized on chitin panicles (24-60 mesh) showed an average activity of lO00 U g- 1. Temperature and pH optima were 60°C and 3"6, respectively. These values were lower than the corresponding ones for the free enzyme (pH 4"4, temperature 65°C). The K m (Michaelis constant) values for soluble starch and hydrolyzed manioc starch were, respectively, 1"25 and 3"94 g litre- i (free enzyme) and 8"6 and 7.8g litre i (immobilized enzyme). Key words: glucoamylase, enzyme immobilization, manioc starch hydrolysis.
INTRODUCTION The production of glucose from starch by enzymatic processes is growing in commercial importance. The use of immobilized glucoamylase in the saccharification step of these processes has some advantages: it enables continuous operation with reduced manpower and reduces enzyme use. However, the use of immobilized enzymes in industrial processes depends on the availability of an active and stable biocatalyst. This note reports the evaluation and characterization of a biocatalyst whose preparation, described in a previous study by Bon et al.,~ was subsequently improved. The literature contains numerous reports on the use of many types of support materials and methods of immobilization for glucoamylase. Table 1 summarizes some of the more recently published works. Chitin, the support material used in this study, has previously been used for immobilization of glucoamylase by Stanley et al. 2 and by Flor and Hayashida. 3 71 Biomass 0144-4565/90/S03.50 © 1990 Elsevier Science Publishers Ltd, England. Printed in Great Britain
72
D. G. Freire, G. L. Sant'Anna Jr
MATERIALS A N D METHODS
Glucoamylase, from Aspergillus niger (AMG Novo L-Novo Industri, Bagsvaerd, Denmark), was immobilized on chitin particles (mean diameter 0.37 mm) activated with hexamethylenediamine and glutaraldehyde following the procedure described by Bon et aL 1 Two types of substrates were used: soluble starch (analytical grade: Reagen) and commercial manioc starch flour, which was enzymatically hydrolyzed with a-amylase (E.C. 3.2.1.1)(Termamyl-Novo Industri, Bagsvaerd, Denmark) using a ratio of 0.1 w/w enzyme/starch. The experimental set-up used for the evaluation of the biocatalyst was as described in a previous paper) 1 The reactor was a small stirred vessel (60 ml) with a removable stainless steel basket (150 mesh) submerged in the liquid. Samples of approximately 40 mg of the biocatalyst particles were put inside the basket and its activity measured as the initial rate of glucose production. One unit of activity was defined as the amount of enzyme which produce 1 pmol of glucose min-~, at 45°C, in buffered (Na acetate 0.1 M) solutions of substrate. Glucose was determined by the glucoseoxydase/peroxydase test (GOD-PAP Merckotest 3395,
TABLE 1 Some Published Works on Glucoamylase Immobilization Support
Half-life
Substrate
Reference
time
Polyethylene vinyl alcohol D E A E - - cellulose
> 150 days (5°C) --
Porous glass fiber Polymethacrylate
8 days --
Corn stover
40 days 105 days (60°C)
Polyvinyl
Imai et al. 4
Soluble starch Maltose
Tomar & Prabhu 5 Toldra et al. 6
(50°C)
(400C)
Polyacrylamide
Maltose
2.5 h (40°C) 60 days (4°C)
Soluble starch Hydrolyzed starch Soluble and hydrolyzed starch Maltose starch
Fiedureck et al. 7 Vallat & Monsan 8 Szajani et aL 9
Imai et aL 10
Character&ation of a glucoamylase immobilized on chitin
73
Darmstadt, Austria). The parameters associated with glucoamylase in solution (free enzyme) were determined using the same methods.
RESULTS AND DISCUSSION The influence of pH on enzyme activity (expressed as relative initial rate of glucose production) is shown in Fig. 1. The immobilized enzyme had an optimum pH value which was lower than that of the free enzyme. This decrease in pH may be related to the existence of positive charges on the surface of the particle, probably associated with residual amino groups. Depending on the ionic strength of the solution, these charges contribute to the establishment of a pH gradient near the particle and thus the immobilized enzyme is submitted to a pH which is different from the pH of the bulk solution. Similar results were observed by Stanley et aL 2 when using chitin activated with glutaraldehyde and by CabraP 2 when using porous silica activated with amine and glutaraldehyde. The influence of temperature on the activity of glucoamylase (free and immobilized) are shown in Fig. 2. The optimum temperature for the immobilized enyzme was 60°C, somewhat lower than the optimum temperature for the free enzyme (65°C). A more rigid molecular structure typical of immobilized enzymes may enhance its sensitivity to temperature inactivation, thus leading to a lower optimum temperature value in comparison with the free enzyme. Other similar results showing differences in optima temperatures for free and immobilized enzymes are reported by Bachler et aL, 13 Maeda and Suzuki, 14 and Szajani et aL 9 The experimental data confined to the ascendant part of the curves shown in Fig. 2 may be represented by an Arrhenius relationship, leading to activation energy values of 12.6 and 6.7 kcal mol -~ for free and relative rote of reaction (%1 100 • @
immobilized enzyme
0 free enzyme
50'
L/ Fig. 1.
Influence of pH on glucoamylase activity.
74
D. G. Freire, G. L. Sant'Anna Jr
immobilized enzymes respectively. A decrease in the activation energy for immobilized enzymes was also reported by Cabral ]2 and Weetal, ]5 who attribute this result to diffusional limitations and conformational effects associated with immobilized biocatalysts. The kinetic parameters of the glucoamylase were determined at 45°C (at the optimal temperature the rate of enzyme inactivation is too high) and optimal pH (4.4 for the free enzyme and 3"6 for the immobilized enzyme). The substrates used were soluble starch and hydrolyzed manioc starch. The experimental data were successfully represented by the classical Michaelis-Menten model. The Lineweaver-Burk plot of these data resulted in straight lines as shown in Figs 3 and 4, with correlation coefficients higher than 0.94. The kinetic parameters obtained from the slope and the intercept of these straight lines are listed in Table 2. From these results, it can be seen that the immobilized enzyme presents higher Km values than the free enzyme. This is related to the increased diffusional resistance and steric effects associated with the immobilized biocatalysts. This same trend was observed by several authors: Park,~6 Nithianandham et al., 17 Tomar and Prabhu, 5 Vallat and Monsan, 8 and Imai et al. 4 The K m value obtained for the free enzyme and soluble starch is close to the values obtained by Kennedy and Epton ~8 and Cabral; ~2 the K m value for the immobilized glucoamylase (8"6 g litre -~ ) is comparable to those reported by CabraP 2 for different supports: celite (5,5 g litre-~), controlled pore glass (8-0 g litre -l) and pumice-stone (6.4 g litre-~). The free enzyme K m value was lower for soluble starch substrate, whose mean molecular weight (MW) is greater than that of the hydrolyzed manioc starch substrate. The decrease of Km with the increase of the substrate MW was also remarked on by Kusunoki et al. ~9and Imai et al. ~o The effect of substrate MW o n K m for immobilized enzymes seems to be less evident and in this experiment a
relative rate of reaction (%) 100
• immobilized enzyme 0 free enzyme
50-
-'// Fig. 2.
:30 40 50 60
~;0 eO
I~emperoture(°c)
Influence of temperature on glucoamylase activity.
Characterization of a glucoamylase immobilized on chitin
75
slight decrease of K m w a s registered with the decrease of substrate MW. This same trend was observed by Vallat and Monsan 8 in experiments with immobilized glucoamylase and two different MW substrates. The VmaX (maximum velocity, Michaelis model) values reported in Table 2
! ~6.0 -- IT
o-FREEENZYME
I- 5.0
~'~
1.0
0.5
i.o
i.5
i
i
i
!
Fig. 3.
.
.
.
.
1/S
(9/IP)-i
z.o i
.
Lineweaver-Burk plot of free and immobilized glucoamylase on soluble starch. S = substrate concentration (g/litre).
TABLE 2 Kinetic Parameters of Immobilized and Free Glucoamylase Enzyme
Free Free
Immobilized Immobilized
Substrate
Soluble starch Hydrolyzed manioc starch Soluble starch Hydrolyzed manioc starch
Km (g litre-/)
Vma× (# mol m i n ¢ g- / support)
Illll ),,
(p mol min- i mg-litre protein)
1.25
56'1
3-94
45"7
8.61
941
7.82
920
76
D. G. Freire, G. L. Sant'Anna Jr
-IT/
~*1
-,..o.,L,zEo
20
.----.--""0""
~........~-
o
" " "
o.1 I
o.2 I
o,~
Fig. 4.
o.a I
l/S (~11)"I r
0.4 |
t/S 10/11 "I
o.1
Lineweaver-Burk plot of free and immobilized glucoamylase on hydrolyzed manioc starch. S = substrate concentration (g/litre).
seem to be little influenced by the substrate MW for the free and immobilized glucoamylase. The biocatalyst used in this work was further tested by Freire 2° under continuous conditions in a laboratory-scale expanded-bed reactor fed with a solution of hydrolyzed manioc starch (15% w/w) at 45°C. A very smooth decrease of the immobilized glucoamylase activity was observed and the experimental data lead to a half-life time of 156 days, a result which is in the range of the best stability data reported in the literature. ACKNOWLEDGEMENT This work was supported by grants of Conselho Nacional de Desenvolvimento Cientffico e Technol6gico, CNPq (Brazil). This support is gratefully acknowledged by the authors. REFERENCES 1. Bon, E., Freire, D. B., Mendes, M. F., Moreira, C. P. & Soares, V. E, Immobilization of glucoamylase on inexpensive supports: A comparative evaluation. Biotechnol. Bioeng., Symposiumno. 14 (1984) 485-92.
Characterization of a glucoamylase immobilized on chitin
77
2. Stanley, W. L., Watters, G. G., Kelly, S. H. & Olson, A. C., Glutaraldehyde immobilized on chitin with glutaraldehyde. Biotechnol. Bioeng., 2t) (1978) 135-40. 3. Flor, P. Q. & Hayashida, S., Production and characteristics of raw-starchdigesting glucoamylase O from a protease -- negative glucosidase negative Aspergillus awanori vat. kaawachi mutant. Applied and Environmental Microb., 45 (3)(1983) 905-12. 4. Imai, K., Shiomi, T., Uchida, K. & Miya, M., Immobilization of enzyme onto polyethylene-vinyl alcohol membrane. Biotechnol. Bioeng., 28 (1986) 198-203. 5. Tomar, M. & Prabhu, K. A., Immobilization of glucoamylase on DEAEcellulose activated with chloride compounds. Enzyme Microb. Technol., 7 (1985) 557-9. 6. Toldra, E, Jansen, N. B. & Tsao, G. T. Use of a porous glass fiber as a support for biocatalyst immobilization. Biotechnol. Letters, $ ( 11 ) (1986) 785-90. 7. Fiedureck, J., Lobarzewski, J., Wojcik, A. & Wolski, T., Optimization of enzyme immobilization on keratin or polyamide-coated bed-shaped polymeric matrix. Biotechnol. Bioeng., 28 (1986) 744-50. 8. Vallat, I. & Monsan, P., Maltodextrin hydrolysis in the fluidized-bed immobilized enzyme reactor. Biotechnol. Bioeng., 23 (1986) 151-9. 9. Szajani, B., Klamar, G. & Ludwig, L., Preparation, characterization and laboratory-scale application of an immobilized glucoamylase. Enzyme Microb. Technol., 7 (1985) 488-92. 10. Imai, K., Shiomi, T., Uchida, K. & Miya, M., Immobilization of enzyme into poly-vinyl-alcohol membrane. Biotechnol. Bioeng., 28 (1986) 1721-6. 11. Xavier, M. T., Soares, V. E, Freire, D. M. G., Moreira, C. P., Mendes, M. E & Bon, E., a-Amylase and glucoamylase immobilized on chitin and ceramic supports. Biomass, 13 (1987) 25-32. 12. Cabral, J. M. S., Estudo de imobilizaqho de enzimas pelo metodo dos metais de transi~o. DSc. thesis, Universidade T6cnica de Lisboa, 1982. 13. Bachler, M. J., Strandberg, G. U. & Similey, K. L., Starch conversion by immobilized glucoamylase. Biotechnol. Bioeng., 12 ( 1970) 85-92. 14. Maeda, J. & Suzuki, H., Preparative of immobilized enzymes by N-vinylpyrrolidone and the general properties of the glucoamylase gel. Biotechnol. Bioeng., 16 (1974) 1517-28. 15. Weetal, H. H., Immobilized enzymes and their application in the food and beverage industry. Process Biochemistry, $ ( 1975 ) 3-30. 16. Park, Y. K., Enzymic properties of fungal amyloglicosidase-resin complex. J. Ferm. Technol., 2 (2)(1974) 140-2. 17. Nithianandham, V. S., Srinivasan, K. S. V., Joseph, K. T. & Santappa, M., Preparation and properties of immobilized amyloglicosidase. Biotechnol. Bioeng., 23 (1982)2273-82. 18. Kennedy, J. F. & Epton, J., Poly N-acryloyl-4 and 5-aminosalicylic acids. Carbohydrate Research, 27 (1973) 11-20. 19. Kusunoki, K., Kawakami, K., Shiraishi, K. & Kai, M., A kinetic expression for hydrolysis of soluble starch by glucoamylase. Biotechnol. Bioeng., 24 (1982) 347-54.
78
D. G. Freire, G. L. Sant'Anna Jr
20. Freire, D. M. G., Imobilizaq~o de amiloglicosidase em quitina. MSc thesis, Escola de Quimica, Universidade Federal do Rio de Janeiro, 1988.
Denise Guimar~es Freire & Geraido Lippel Sant'Anna Jr* COPPE, Escola de Quimica and Instituto de Quimica, Universidade Federal do Rio de Janeiro, PO Box 68502, 21945 Rio de Jane&o, Brazil (Received 19 April 1989; accepted 26 June 1989) *To whom correspondence should be addressed.