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Thermostable 1-amylase production by Bacillus licheniformis cells immobilized on polyacrylates with cyclic carbonate groups in the side chain
Microbiol. Res. (1998) 153, 157-162
©
Gustav Fischer Verlag
Thermostable a-amylase production by Bacillus licheniformis cells immobilized on polyacrylates with cyclic carbonate groups in the side chain E. Dobreva, V. Ivanova, M. Stefanova, A. Tonkova, L. Kabaivanova, D. Spassova Institute of Microbiology, BulgarianAcademy of Sciences, 26Acad. G. Bonchev str., 1113 Sofia, Bulgaria Telephone:+ 359-2713 -31-63, fax:+359-2-700 109 Accepted: January 22, 1998
Abstract A considerable increase (up to 35%) of thermostable a -amylase yield was achieved by Bacillus licheniformis cells, immobilized on polymers containing cyclic carbonate groups in comparison to the synthesis by free cells. The increase of enzyme production was dependent on the manner of cell immobilization and on thekind of polymers used. Thea-amylase yield by immobilized "growing" cells was higher than that reached by immobilized "washed" cells. The homopolymer poly [(2-oxo-1 ,3-dioxolan-4-yl) methyl 2-propenoate] and especiallyits methyl derivative were found to be more suitable for bacterial cell immobilization than the copolymer N-vinylpyrrolydone poly [(2-oxo-1,3-dioxolan-4-yl) methyl 2-propenoate]. After 576h (4 cycles) of repeated batchfermentations, all biocatalysts used showed approximately equal a-amylase activity (2000 U/ml), correspondi ng to 60-80% residual activity depending on the kindof the biocatalyst. Key words: Immobilization - Bacillus licheniformis - thermostable a-amylase-polymers with cyclic carbonate groups
Introduction The simplest immobilization procedure for biological substances consists of their direct contact with preformed reactive polymers, such as maleic or methacrylic anhydride based copolymers (Levin et al . 1964; Zingaro and Uziel 1970; Zaborsky 1973). However these supports were synthesized in nonaqueous solvents and decompose in water. In their turn, polymers containing cyCorresponding author: E. Dobreva
clic carbonate groups would have been of interest as carriers because of the high reactivity and selectivity of dioxolan ring towards aminogroups. These assumptions were based on the successful applications of cyclic carbonate derivatives of cellulose (Barker et al. 1971) or dextran (Doane et al . 1968) for immobilization of different enzymes such as ~-gluco sid ase, trypsin, dextranase (Kennedy and Zamir 1973 ; Cheetham and Richards 1973). However these supports had a rather short spacer and the toxic ethyl-chloroformiate was used in their synthesis. Polymers with a cyclic carbonate in the side chain had been obtained after a radical polymerization of acrylic monomers (Couvert and Bross 1990) but to our knowledge there are no studies on their use for immobilization of biological materials. The purpose of this study was to investigate the possibilities for thermostable a -amylase production by Bacillu s licheniformis cells immobilized on polyacrylates with 1,3-dioxolan-2-on (cyclic carbonate) side group.
Materials and methods Microorgani sm and media. Bacillus licheniformis 44 MB 82 G strain, resistant to catabolite repression, producer of thermostable a -amylase was used (Tonkova et al. 1989; Tonkova 1991). Nutrient broth supplemented with 1% (w/v) soluble starch and 2% (w/v) glucose was used as seed medium. The fermentation medium contained [in % (w/v)]: glucose-6.0; beef extract "LabLemco powder"-1.5; peptone-1.5; K2HPOc 1.04; corn steep liquor-0.66; CaCI2- O.I I ; pH 7.0-7.2. Microbiol. Res. 153 (1998) 2
157
Immobilization procedures. Homopolymers: Poly [(2-
Scanning electron microscopy (SEM). Biocatalyst's par-
oxo-I,3-dioxolan-4-yl) methyl 2-propenoate]-1 or (2)poly [(2-oxo-I,3-dioxolan-4-yl) methyl 2-methyl-2propenoate]-2 and the copolymer N-vinyl-pyrrolydone poly [(2-oxo-1,3-dioxolan-4-yl) methyl 2-propenoate]3, obtained according to the procedure of Kossev and Troev (1993) were used as carriers. The binding of bacterial cells on them was carried out according to scheme 1 and their practical realization was done in two manners:
ticles were fixed for 2 h in 2% (wIv) glutaraldehyde. After washing with saline solution, they were dehydrated in 30-100% water ethanol series. The air-dried particles were coated with 120-130 A of gold in argon medium in an Edwards apparatus (model 150 A). The SEM observations were done on a scanning device attached to a Zeiss electron microscope (model 10 C) at 20 kV accelerating voltage with a 5 to 6 nm electron beam.
i. Immobilization of "growing" cells-O.4g of U'V-sterilized polymers and growing bacterial cells were cultivated in seed medium for 18h, 40°C on a shaker (220 rpm). Biocatalysts obtained were separated and washed with sterile water and 0.066 M phosphate buffer (pH 6.5) to eleminate the adsorbed cells. ii. Immobilization of "washed" cells - cells of late exponential phase, grown in seed medium were harvested by centrifugation (4000 rpm, 20min, 4°C). They were washed twice with saline solution and after centrifugation the wet biomass (0.2 g) was placed in 25 ml 0.66 M phosphate buffer (pH 6.5), containing 0.4 g of UV-sterilized polymers for 6 h at 25°C with stirring. Then the biocatalysts obtained were washed with sterile water and phosphate buffer.
a-amylase synthesis. The biocatalysts obtained were transferred into 50 ml fermentation medium and cultivated at 40°C on a rotary shaker (240 rpm). The enzyme activity was measured every 24 h from 72 up to 144 h of cultivation. Parallel experiments were also run with free bacterial cells. The repeated batch fermentations were performed with washed biocatalysts from the previous cycle, reintroduced into fresh medium. The duration of each cycle was 144 h. a-amylaseactivity was assayed according to the method of Pantschev et at. (1981). One unit of enzyme activity was defined as the amount of enzyme that catalysed the hydrolysis of 0.162 mg soluble starch to dextrins per min at 30°C, pH 6.5.
P-C-OCH 2-CH-CH2 +
o
b o 'c
Where P is:
(1)
Results The results for a-amylase production with immobilized on homopolymer (1), copolymer (3) or free B. licheniformis cells (Table 1) showed that almost all of the biocatalysts obtained were more effective enzyme producers than free cells (increase from 5 to 21% in a-amylase yield) with the exception of the copolymer biocatalyst, containing washed bacterial cells, where a decrease of 10% in a-amylase yield was observed. In general the enzyme production by immobilized on copolymer cells was 10-15% lower than that with immobilized on homopolymer (1) cells. With regard to immobilization procedure (with "growing" or "washed" cells), the use of growing cells turned out to be more suitable. In this case an increase from 14 to 22% in comparison to "washed" cells was observed. The enhanced enzyme yield (21-23%) obtained with immobilized "growing" cells on homopolymer (1) suggested a comparative investigation with its methyl derivative [homopolymer (2)]. An increase in a-amylase production of 10% compared to homopolymer (1) and 35% compared to free cells was achieved (Fig. 1). The SEM observations of this biocatalyst (Fig. 2) at the end of the fermentation process (144 h) showed that cells were attached to the surface of the po1yacry1ates (Fig. 2A) and also entrapped inside the polymer network (Fig. 2B). Fig. 2C illustrates cell walls of lyzed bacterial cells bound to the polymer.
H2N-Cell~P-C-OCH2-CH-CH2-0CHN-
0
OH
0
Cell
6
(2)
Scheme 1. 158
Microbial. Res. 153 (1998) 2
Table 1. Dynamics of a-amylase synthesis by free or polymer immobilized cells of B. licheniformis. Batch cultivation with
Cells used in the immobilization process
a-amylase production in the course of cultivation 96h
72h
120h
144h
D/ml
%*
D/ml
%
D/ml
%
D/ml
%
immobilized on homopolymer (1) cells
growing washed
2000 1750
121 106
2400 2100
120 105
2900 2500
123 106
3400 3000
121 107
immobilized on copolymer (3) cells
growing washed
1800 1500
109 91
2200 1800
110 90
2600 2100
III 89
3100 2500
110 90
1650
100
2000
100
2350
100
2800
100
free cells (control)
* - % of the control assumed as 100
Fig. 1. Production of thermostable a-amylase with free (0-0) or immobilized on homopolyO + - - - - - - - + - - - - - - - + - - - - - - - - - 1 r - - - - - - - - i mers (1) (6-6) and (2) (0-0) 131 72 96 144 "growing" cells of B. licheniformis TIIte [h]
With regard to operational stability of the different biocatalysts used (Fig. 3), repeated batch fermentations with biocatalysts containing "growing" cells showed 40% loss of their initial activity at the end of the 4 th cultivation cycle, while those with "washed" cells retained about 80% of the enzyme activity at the same conditions. At the end of the process (576 h) all biocatalysts used maintained the same level of activity (about 2000U/ml).
Discussion The difficulties related to immobilization of living cells consist in the requirement of very mild conditions and procedures, including suitable carriers and in the accumulation of biomass due to continuous reproduction. The activity, efficiency and operational stability of the biocatalyst obtained are the most important for their quality (Kobayashi et al. 1980; Klein and Vorlop 1983).
The interrelation of these three factors determined the possibilities of preference of one of it depending on the concrete case. The efficiency of thermostable a-amylase production by Bacillus licheniformis cells immobilized on polyacrylates with cyclic carbonate as side group was found to be dependent on the type of polymer (homo- or co-) and on the kind of cells used for immobilization. The activity of homopolymer biocatalysts was in all cases higher than that of the copolymer ones. The reason for that probably consists in the lower quantity of immobilized cells because of the less frequency of cyclic carbonate groups in the copolymer. The more effective immobilization of "growing" cells in comparison to "washed" cells confirmed our previous investigations. Similar results were obtained with Bacillus licheniformis cells immobilized on acrylonitrile-acrylamide membranes (Tonkova et al. 1994, Ivanova et al. 1995). The probable reason for that was the greater viability of the "growing" cells at immobilization. May be this fact Microbiol. Res. 153 (1998) 2
159
8
I----f .
Fig.2. Scanning electron micrographs of bacterial cells immobilized on homopolymer (2) after 144h fermentation. A-attached to the polymer; B-entrapped in the polymer; C-lyzed cells; Scale bar =0.5!tm 160
Microbiol. Res. 153 (1998) 2
4000
-...... ='
E
:::J
..
~
"> o
immobilized "growing"cells on hom opolym er( 2)
3500 3000 2500
• immobilized "growing" cell s on homopolymer (1)
2000
III
ell
1500
~ e
1000
E
W
o immobilized "growing" cells on copolym er (3)
500 0 3
2
4
. im m obilized "w as hed" cells on homopolymer (1)
Cycles • immobilized "washed" cell s on copo lym er (3)
Fig.3. Ope rational stab ility of the biocatalysts.
accounts for the higher initial activity of the biocatal ysts obtained with this kind of cells. On the other hand the immobili zation of "washed" cells was a longer procedure which perhap s moderated cell viability but made the bonds with polymers stronger. That is why the biocatalysts with this kind of cells showed lower initial enzyme activity but higher operational stability compared to "growing" cells. The data presented showed the possibility of preparing biocatalysts where enzyme activity and operational stability were balanced in a way to ensure a suitable product from economic viewpoint.
Conclusion The investigated polymers, containing cyclic carbon ate groups appeared to be suitable carriers for bacterial cell immobilization. An optimization of immobilization conditions and a following use of the biocatalysts obtained in semicontinuous or continuous processes would make them more effective and perspective.
Acknowledgement Thi s work was supported by a Project grant K 24 from the National Scienti fic Foundation, Bulgarian Ministry of Scien ce and Edu cation . We also thank Dr. Troev from the Institute of polymers and K. Kossev from the Institute of Applied Min eralogy of the Bulgarian Academy of Sciences for kind ly supplying the polyacrylates.
References Barker, S. A., Tun , H. c, Doss, S. H., Gray, Ch. 1., Kennedy, 1. F. ( 197 1): Preparation of cellulose car bonate. Carbohydr. Res. 17 (2), 471- 474. Cheetham, N. W. H., Rich ards, G. N. (1973 ): Dextranases III. Insolubilization of a bacterial de xtranase. Carbohydr. Res. 30 (I), 99-107. Couvert, D., Brosse, 1. C. (1990) : Monomers acr yliques a function carbonate cyclique 2. Modification chemique de copolymeres a groupements carbonate cyclique later aux . Makromoi. Chem. 191, 1311-13 19. Doane, W. M., Shasha, B. S., Stout, E. I., Russell, Ch. R., Rist, C. E. ( 1968) : Reaction of starch with carbohydrate tran scarbo nates. Carbohydr. Res. 8 (3), 266-274. Ivanova, v., Stefanova, M., Tonkova, A., Dobre va, E., Spassoya, D. ( 1995): Screening of a growi ng cell immobilizatio n proce dure for the biosynth esis of thermostable a -amylases . Appl. Biochem . Biotechnol. 50 , 305-3 16. Kenn edy. Y F., Zam ir, A. (1973) : Use of cell ulose carbonate for the insolubilization of enzymes. Carbohydr. Res. 29 (2), 497- 50 1. Klein, Y , Vorlop, K. D. (\983) : Imm ob ilized cell s. Catalyst preparation and reaction performance. ACS Symp. Ser. 207/l 2, 377- 392. Kobayashi, T., Katagiri, K., Ohm iya, K., Shimizu, S. (1980): Effect of mass transfer on operatio nal stability of immobilized enzy me. 1. Ferm ent. Techn ol. 58 , 23- 31. Kossev, K., Troev, K: (1993) : Hom o- and copolymers containing 1,3-di oxolan- 2-on gro up, 1. Synthesis of polymer carriers. XI. Symposium " Polymers 93" Bulgaria. Lev in, Y., Pecht, M., Goldstein, L., Katchalski, E. (\964): A water-insoluble polyanionic der ivative of trypsin. Biochemistry 3, 1905 -1909. Microbiol. Res. 153 (1998) 2
16 I
Pantschev, c. Klenz, G., Hafner, B. (1981): Vergleichende Charakterisierung von n-Amylasepraparaten. Lebensrnittelindustrie 28, 71-74. Tonkova, A. (1991): Effect of glucose and citrate on a-amylase production in B. licheniformis. J. Basic Microbiol. 31, 217-222. Tonkova, A., Emanuilova, E. (1989): Method for obtaining of thermostable a-amylase. Bulg. Patent N 88845. Tonkova, A., Ivanova, v., Dobreva, E., Stefanova, M., Spas-
162
Microbiol. Res. 153 (1998) 2
so va, D. (1994): Thermostable a-amylase production by immobilized B. licheniformis cells in agar gel and on acrylonitrile acrylamide membranes. Appl. Microbiol. Biotechnol. 41, 517-522. Zaborsky, O. R. (1973): Immobilized enzymes. CRC Press Cleveland, Ohio, Ed. R. C. Weast. Zingaro, R. A., Uziel, M. (1970): Preparation and properties of active, insoluble alkaline phosphatase. Acta Biochem. Biophys.213,371-380.
©
Gustav Fischer Verlag
Thermostable a-amylase production by Bacillus licheniformis cells immobilized on polyacrylates with cyclic carbonate groups in the side chain E. Dobreva, V. Ivanova, M. Stefanova, A. Tonkova, L. Kabaivanova, D. Spassova Institute of Microbiology, BulgarianAcademy of Sciences, 26Acad. G. Bonchev str., 1113 Sofia, Bulgaria Telephone:+ 359-2713 -31-63, fax:+359-2-700 109 Accepted: January 22, 1998
Abstract A considerable increase (up to 35%) of thermostable a -amylase yield was achieved by Bacillus licheniformis cells, immobilized on polymers containing cyclic carbonate groups in comparison to the synthesis by free cells. The increase of enzyme production was dependent on the manner of cell immobilization and on thekind of polymers used. Thea-amylase yield by immobilized "growing" cells was higher than that reached by immobilized "washed" cells. The homopolymer poly [(2-oxo-1 ,3-dioxolan-4-yl) methyl 2-propenoate] and especiallyits methyl derivative were found to be more suitable for bacterial cell immobilization than the copolymer N-vinylpyrrolydone poly [(2-oxo-1,3-dioxolan-4-yl) methyl 2-propenoate]. After 576h (4 cycles) of repeated batchfermentations, all biocatalysts used showed approximately equal a-amylase activity (2000 U/ml), correspondi ng to 60-80% residual activity depending on the kindof the biocatalyst. Key words: Immobilization - Bacillus licheniformis - thermostable a-amylase-polymers with cyclic carbonate groups
Introduction The simplest immobilization procedure for biological substances consists of their direct contact with preformed reactive polymers, such as maleic or methacrylic anhydride based copolymers (Levin et al . 1964; Zingaro and Uziel 1970; Zaborsky 1973). However these supports were synthesized in nonaqueous solvents and decompose in water. In their turn, polymers containing cyCorresponding author: E. Dobreva
clic carbonate groups would have been of interest as carriers because of the high reactivity and selectivity of dioxolan ring towards aminogroups. These assumptions were based on the successful applications of cyclic carbonate derivatives of cellulose (Barker et al. 1971) or dextran (Doane et al . 1968) for immobilization of different enzymes such as ~-gluco sid ase, trypsin, dextranase (Kennedy and Zamir 1973 ; Cheetham and Richards 1973). However these supports had a rather short spacer and the toxic ethyl-chloroformiate was used in their synthesis. Polymers with a cyclic carbonate in the side chain had been obtained after a radical polymerization of acrylic monomers (Couvert and Bross 1990) but to our knowledge there are no studies on their use for immobilization of biological materials. The purpose of this study was to investigate the possibilities for thermostable a -amylase production by Bacillu s licheniformis cells immobilized on polyacrylates with 1,3-dioxolan-2-on (cyclic carbonate) side group.
Materials and methods Microorgani sm and media. Bacillus licheniformis 44 MB 82 G strain, resistant to catabolite repression, producer of thermostable a -amylase was used (Tonkova et al. 1989; Tonkova 1991). Nutrient broth supplemented with 1% (w/v) soluble starch and 2% (w/v) glucose was used as seed medium. The fermentation medium contained [in % (w/v)]: glucose-6.0; beef extract "LabLemco powder"-1.5; peptone-1.5; K2HPOc 1.04; corn steep liquor-0.66; CaCI2- O.I I ; pH 7.0-7.2. Microbiol. Res. 153 (1998) 2
157
Immobilization procedures. Homopolymers: Poly [(2-
Scanning electron microscopy (SEM). Biocatalyst's par-
oxo-I,3-dioxolan-4-yl) methyl 2-propenoate]-1 or (2)poly [(2-oxo-I,3-dioxolan-4-yl) methyl 2-methyl-2propenoate]-2 and the copolymer N-vinyl-pyrrolydone poly [(2-oxo-1,3-dioxolan-4-yl) methyl 2-propenoate]3, obtained according to the procedure of Kossev and Troev (1993) were used as carriers. The binding of bacterial cells on them was carried out according to scheme 1 and their practical realization was done in two manners:
ticles were fixed for 2 h in 2% (wIv) glutaraldehyde. After washing with saline solution, they were dehydrated in 30-100% water ethanol series. The air-dried particles were coated with 120-130 A of gold in argon medium in an Edwards apparatus (model 150 A). The SEM observations were done on a scanning device attached to a Zeiss electron microscope (model 10 C) at 20 kV accelerating voltage with a 5 to 6 nm electron beam.
i. Immobilization of "growing" cells-O.4g of U'V-sterilized polymers and growing bacterial cells were cultivated in seed medium for 18h, 40°C on a shaker (220 rpm). Biocatalysts obtained were separated and washed with sterile water and 0.066 M phosphate buffer (pH 6.5) to eleminate the adsorbed cells. ii. Immobilization of "washed" cells - cells of late exponential phase, grown in seed medium were harvested by centrifugation (4000 rpm, 20min, 4°C). They were washed twice with saline solution and after centrifugation the wet biomass (0.2 g) was placed in 25 ml 0.66 M phosphate buffer (pH 6.5), containing 0.4 g of UV-sterilized polymers for 6 h at 25°C with stirring. Then the biocatalysts obtained were washed with sterile water and phosphate buffer.
a-amylase synthesis. The biocatalysts obtained were transferred into 50 ml fermentation medium and cultivated at 40°C on a rotary shaker (240 rpm). The enzyme activity was measured every 24 h from 72 up to 144 h of cultivation. Parallel experiments were also run with free bacterial cells. The repeated batch fermentations were performed with washed biocatalysts from the previous cycle, reintroduced into fresh medium. The duration of each cycle was 144 h. a-amylaseactivity was assayed according to the method of Pantschev et at. (1981). One unit of enzyme activity was defined as the amount of enzyme that catalysed the hydrolysis of 0.162 mg soluble starch to dextrins per min at 30°C, pH 6.5.
P-C-OCH 2-CH-CH2 +
o
b o 'c
Where P is:
(1)
Results The results for a-amylase production with immobilized on homopolymer (1), copolymer (3) or free B. licheniformis cells (Table 1) showed that almost all of the biocatalysts obtained were more effective enzyme producers than free cells (increase from 5 to 21% in a-amylase yield) with the exception of the copolymer biocatalyst, containing washed bacterial cells, where a decrease of 10% in a-amylase yield was observed. In general the enzyme production by immobilized on copolymer cells was 10-15% lower than that with immobilized on homopolymer (1) cells. With regard to immobilization procedure (with "growing" or "washed" cells), the use of growing cells turned out to be more suitable. In this case an increase from 14 to 22% in comparison to "washed" cells was observed. The enhanced enzyme yield (21-23%) obtained with immobilized "growing" cells on homopolymer (1) suggested a comparative investigation with its methyl derivative [homopolymer (2)]. An increase in a-amylase production of 10% compared to homopolymer (1) and 35% compared to free cells was achieved (Fig. 1). The SEM observations of this biocatalyst (Fig. 2) at the end of the fermentation process (144 h) showed that cells were attached to the surface of the po1yacry1ates (Fig. 2A) and also entrapped inside the polymer network (Fig. 2B). Fig. 2C illustrates cell walls of lyzed bacterial cells bound to the polymer.
H2N-Cell~P-C-OCH2-CH-CH2-0CHN-
0
OH
0
Cell
6
(2)
Scheme 1. 158
Microbial. Res. 153 (1998) 2
Table 1. Dynamics of a-amylase synthesis by free or polymer immobilized cells of B. licheniformis. Batch cultivation with
Cells used in the immobilization process
a-amylase production in the course of cultivation 96h
72h
120h
144h
D/ml
%*
D/ml
%
D/ml
%
D/ml
%
immobilized on homopolymer (1) cells
growing washed
2000 1750
121 106
2400 2100
120 105
2900 2500
123 106
3400 3000
121 107
immobilized on copolymer (3) cells
growing washed
1800 1500
109 91
2200 1800
110 90
2600 2100
III 89
3100 2500
110 90
1650
100
2000
100
2350
100
2800
100
free cells (control)
* - % of the control assumed as 100
Fig. 1. Production of thermostable a-amylase with free (0-0) or immobilized on homopolyO + - - - - - - - + - - - - - - - + - - - - - - - - - 1 r - - - - - - - - i mers (1) (6-6) and (2) (0-0) 131 72 96 144 "growing" cells of B. licheniformis TIIte [h]
With regard to operational stability of the different biocatalysts used (Fig. 3), repeated batch fermentations with biocatalysts containing "growing" cells showed 40% loss of their initial activity at the end of the 4 th cultivation cycle, while those with "washed" cells retained about 80% of the enzyme activity at the same conditions. At the end of the process (576 h) all biocatalysts used maintained the same level of activity (about 2000U/ml).
Discussion The difficulties related to immobilization of living cells consist in the requirement of very mild conditions and procedures, including suitable carriers and in the accumulation of biomass due to continuous reproduction. The activity, efficiency and operational stability of the biocatalyst obtained are the most important for their quality (Kobayashi et al. 1980; Klein and Vorlop 1983).
The interrelation of these three factors determined the possibilities of preference of one of it depending on the concrete case. The efficiency of thermostable a-amylase production by Bacillus licheniformis cells immobilized on polyacrylates with cyclic carbonate as side group was found to be dependent on the type of polymer (homo- or co-) and on the kind of cells used for immobilization. The activity of homopolymer biocatalysts was in all cases higher than that of the copolymer ones. The reason for that probably consists in the lower quantity of immobilized cells because of the less frequency of cyclic carbonate groups in the copolymer. The more effective immobilization of "growing" cells in comparison to "washed" cells confirmed our previous investigations. Similar results were obtained with Bacillus licheniformis cells immobilized on acrylonitrile-acrylamide membranes (Tonkova et al. 1994, Ivanova et al. 1995). The probable reason for that was the greater viability of the "growing" cells at immobilization. May be this fact Microbiol. Res. 153 (1998) 2
159
8
I----f .
Fig.2. Scanning electron micrographs of bacterial cells immobilized on homopolymer (2) after 144h fermentation. A-attached to the polymer; B-entrapped in the polymer; C-lyzed cells; Scale bar =0.5!tm 160
Microbiol. Res. 153 (1998) 2
4000
-...... ='
E
:::J
..
~
"> o
immobilized "growing"cells on hom opolym er( 2)
3500 3000 2500
• immobilized "growing" cell s on homopolymer (1)
2000
III
ell
1500
~ e
1000
E
W
o immobilized "growing" cells on copolym er (3)
500 0 3
2
4
. im m obilized "w as hed" cells on homopolymer (1)
Cycles • immobilized "washed" cell s on copo lym er (3)
Fig.3. Ope rational stab ility of the biocatalysts.
accounts for the higher initial activity of the biocatal ysts obtained with this kind of cells. On the other hand the immobili zation of "washed" cells was a longer procedure which perhap s moderated cell viability but made the bonds with polymers stronger. That is why the biocatalysts with this kind of cells showed lower initial enzyme activity but higher operational stability compared to "growing" cells. The data presented showed the possibility of preparing biocatalysts where enzyme activity and operational stability were balanced in a way to ensure a suitable product from economic viewpoint.
Conclusion The investigated polymers, containing cyclic carbon ate groups appeared to be suitable carriers for bacterial cell immobilization. An optimization of immobilization conditions and a following use of the biocatalysts obtained in semicontinuous or continuous processes would make them more effective and perspective.
Acknowledgement Thi s work was supported by a Project grant K 24 from the National Scienti fic Foundation, Bulgarian Ministry of Scien ce and Edu cation . We also thank Dr. Troev from the Institute of polymers and K. Kossev from the Institute of Applied Min eralogy of the Bulgarian Academy of Sciences for kind ly supplying the polyacrylates.
References Barker, S. A., Tun , H. c, Doss, S. H., Gray, Ch. 1., Kennedy, 1. F. ( 197 1): Preparation of cellulose car bonate. Carbohydr. Res. 17 (2), 471- 474. Cheetham, N. W. H., Rich ards, G. N. (1973 ): Dextranases III. Insolubilization of a bacterial de xtranase. Carbohydr. Res. 30 (I), 99-107. Couvert, D., Brosse, 1. C. (1990) : Monomers acr yliques a function carbonate cyclique 2. Modification chemique de copolymeres a groupements carbonate cyclique later aux . Makromoi. Chem. 191, 1311-13 19. Doane, W. M., Shasha, B. S., Stout, E. I., Russell, Ch. R., Rist, C. E. ( 1968) : Reaction of starch with carbohydrate tran scarbo nates. Carbohydr. Res. 8 (3), 266-274. Ivanova, v., Stefanova, M., Tonkova, A., Dobre va, E., Spassoya, D. ( 1995): Screening of a growi ng cell immobilizatio n proce dure for the biosynth esis of thermostable a -amylases . Appl. Biochem . Biotechnol. 50 , 305-3 16. Kenn edy. Y F., Zam ir, A. (1973) : Use of cell ulose carbonate for the insolubilization of enzymes. Carbohydr. Res. 29 (2), 497- 50 1. Klein, Y , Vorlop, K. D. (\983) : Imm ob ilized cell s. Catalyst preparation and reaction performance. ACS Symp. Ser. 207/l 2, 377- 392. Kobayashi, T., Katagiri, K., Ohm iya, K., Shimizu, S. (1980): Effect of mass transfer on operatio nal stability of immobilized enzy me. 1. Ferm ent. Techn ol. 58 , 23- 31. Kossev, K., Troev, K: (1993) : Hom o- and copolymers containing 1,3-di oxolan- 2-on gro up, 1. Synthesis of polymer carriers. XI. Symposium " Polymers 93" Bulgaria. Lev in, Y., Pecht, M., Goldstein, L., Katchalski, E. (\964): A water-insoluble polyanionic der ivative of trypsin. Biochemistry 3, 1905 -1909. Microbiol. Res. 153 (1998) 2
16 I
Pantschev, c. Klenz, G., Hafner, B. (1981): Vergleichende Charakterisierung von n-Amylasepraparaten. Lebensrnittelindustrie 28, 71-74. Tonkova, A. (1991): Effect of glucose and citrate on a-amylase production in B. licheniformis. J. Basic Microbiol. 31, 217-222. Tonkova, A., Emanuilova, E. (1989): Method for obtaining of thermostable a-amylase. Bulg. Patent N 88845. Tonkova, A., Ivanova, v., Dobreva, E., Stefanova, M., Spas-
162
Microbiol. Res. 153 (1998) 2
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