a new method for immobilization of microbial cells by cross-linking
TRANSCRIPT
A p p l i e d Biochemistry and Microbiology, Vol. 36, No. 3, 2000, pp. 310-314. Translated from Prikladnaya Biokhimiya t Mikrobiologiya, Vol. 36, No. 3, 2000, pp. 359-364. riginal Russian Text Copyright �9 2000 by Abelyan.
A New Method for Immobilization of Microbial Cells by Cross-Linking
V. A. Abelyan
Institute of Microbiology, National Academy of Sciences of Armenia, Abovyan, 378510 Armenia
Received March 12, 1998
Abstraet--A method for immobilization of microbial cells was designed. The method uses generation of reac- tive aldehyde groups on the cell wall surface under conditions of periodate oxidation. The linking of aldehyde groups by various bifuctional aromatic diamines and then by glutaraldehyde produced immobilized cells, which are promising for use in biocatalysis with high-molecular-weight substrates.
Immobilized cells (IC) and immobilized enzymes (IE) currently attract attention in many areas of science and technology because of their great importance to both theory and practice [1-3]. The use of extreme- philic microorganisms seems to be a special and very promising branch. Thus, the use of thermophilic micro- organisms or their enzymes requires the use of heat- resistant carders and materials. Although this line seems promising for the use of 1t2 and IE, it is poorly studied and the immobilization approaches available are mainly unsuitable for extremephilic organisms. Studies of stabilization and immobilization approaches based on the cell wall structure and specific features of microorganisms are of special interest because they are promising for production of biocatalysts of high activ- ity and stability under extreme conditions. The stabili- zation of the necessary enzymatic activity of cells under conditions of their arrested growth or the mainte- nance of such activity of viable cells under conditions of their minimal growth in the flow are the main requirements for successful uses of IC and IE.
In this work, a new method for cell immobilization is proposed, which can be used for both mesophilic and thermophilic microorganisms.
MATERIALS AND METHODS
In this work, we used aspartase producers (Erwinia aroidea TSMPM V-2970 and Bacillus subtilis TSMPM V-2597 [4]), producers of L-aspartate-13-decarboxylase (Pseudomonas sp. INMIA-1455 and Alcaligenesfaecalis TSMPM V-3197 [5]), and the producer of fumarase Brevibacterium sp. INMIA-783 [6].
Aspartase, L-aspartate-13-decarboxylase, and fuma- rase were assayed as described in [7], [8], and [9], respectively. L-Aspartic, L-malic, and fumaric acids, and alanine were from Sigma (USA); glutaraldehyde was from Reanal (Hungary); and other reagents were
domestic products of chemical purity grade. Diamines and dihydrazides were synthesized at the Department of Organic Chemistry, Yerevan State University.
RESULTS AND DISCUSSION
Microbial cells were pretreated with sodium perio- date, and then aldehyde groups generated on the cell wall surface were linked with the bifunetional reagent N,N'-m-phenylene diasparimide (PDAI), Such treat- ment resulted in preparations displaying a higher enzy- matic activity in comparison to initial cells.
Microbial cell walls contain polymeric carbohydrates. Sodium periodate opens the carbohydrate rings and exposes free aldehyde groups tO the cell wall surface. Moreover, this pretreatment significantly increases the cell permeability, resulting, on average, in a three to fourfold increase in the specific enzymatic activity. Treatment with PDAI resulted in the linkage of alde- hyde groups, which caused an additional increase in the cell permeability and transferred the cells to a steady state, which also significantly increased their stability.
The treatment of all cell strains studied was optimal when a 0.05% solution of sodium periodate in distilled water was used (Table 1). The enzymatic activity of all producers was found to increase significantly in com- parison to the initial activity. The use of 0.1 to 2.0 ml of 0.05% sodium periodate per g wet cell biomass or from 0.12 to 9.3 Bmol of sodium periodate per g biomass (optimal 0.25 ml) was efficient (Table 2).
The optimal time of cell treatment for all producers was from 15 to 90 min; the maximum activity of aspar- tate and fumarate producers was found after 60 min of the cell treatment, and the maximum activity of L- aspartate-13-decarboxylase producers was found after 45 min of treatment.
The effective temperature of cell treatment was 3- 7~ with the optimum at 5~ for producers of aspartase
0003-6838/00/3603-0310525.00 �9 2000 MAIK "Nauka/Interperiodica"
A NEW METHOD FOR IMMOBILIZATION OF MICROBIAL CELLS
Table 1. Dependence of the activity of microbial cells on the concentration of sodium periodate
311
Culture
E. aroidea
B. subtilis
A. faecalis
Pseudomonas sp. Brevibacterium sp.
Time of treatment, min
60
60 45 45 60
Activity of biomass, mmol/g wet cells per h
concentration 0.5 ml of NaIO 4, 5~ %
0 0.025 0.05 0.1 0.2
1.0 2.1 0.8
2.4 9.3
2.1
2.5 1.9
9.4 18.1
2.5
3.5 2.2
10.0 25.0
1.7
2.8 2.0
9.9 17.3
0.9
0.9 1.8
8.8 6.5
Table 2. Dependence of the activity of microbial cells on the volume of a 0.05% solution of sodium periodate
Culture
E. aroidea
B. subtilis
A. faecalis
Pseudomonas sp. Brevibacterium sp.
Time of treatment, min
60
60 45 45
60
Activity of biomass, mmol/g wet cells per h
volume of sodium periodate, 5~ ml
0 0.10 1.0 2.0
1.0 2.0
0.7 2.4
9.3
1.8
2.8 1.7
12.6
17.7
0.25 0.5
3.0 2.5
6.0 3.5 1.9 1.9
14.2 11.7 28.7 25.1
1.5
2.5
2.9 10.5 16.9
1.5 1.6
1.4 9.4
8.1
Table 3. Dependence of the activity of microbial cells on the time of treatment with sodium periodate (1 ml of 0.05% solution per g wet cells ofA. ~aecalis and 0.25 ml of 0.25% solution in other cases, 5~
Activity of the biomass, mmol/g wet cells per h
Culture time of treatment, min
0 15 30 45 60 90
E. aroidea
B. subtilis
A. faecalis
Pseudomonas sp.
Brevibacterium sp.
1.0
2.1
0.8
2.4
9.3
2.2
2.2
2.2
19.5
16.9
2.2
4.6
3.5
20.6
19.2
2.5
3.5
3.7
25.5
23.0
3.0
6.0
2.7
22.5
28.9
2.2
1.9
2.4
18.8
20.3
and fumarase and 3-10~ with the same optimum at 5~ for producers of L-aspartate-13-decarboxylase (Table 4).
Thus, these results allowed us to recommend 0.25 ml of 0.05% of sodium periodate for the treatment of 1 g of wet cells of E. aroidea, B. subtilis, Pseudomo: nas sp., and Brevibacterium sp. at 5~ 1.0 ml of 0.05% of sodium periodate is recommended for treatment of A. faecalis at 5~ Producers of aspartase and fumarase should be treated for 60 min, and producers of L-aspar- tate-I~-fumarase for 45 min.
After the cell treatment with sodium periodate, 0.3 ml of ethylene glycol was added per ml solution (0.05 ml per lamol sodium periodate) and the sample
was kept at room temperature in the dark for 15-20 min until complete degradation of excess sodium periodate and termination of the reaction.
The cells treated with sodium periodate were sepa- rated by centrifugation (4000 g for 15-20 min), sus- pended in PDAI solution in buffer, and kept at room temperature for 10-24 h.
The optimal concentration of PDAI for all cultures studied was 0.1%, but the linking was also successful at concentrations of 0.05 to 1.0% (Table 5). The optimal volume of 0.1% PDAI was 0.25 ml, but volumes rang- ing from 0.1 to 2.0 ml or (0.16--4.0) • 10 -3 lamol PDAI per g biomass were also efficient (Table 6). The opti- mum pH of PDAI solution, which provided the highest
APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 36 No. 3 2000
312 ABELYAN
Table 4. Dependence of the activity of microbial cells on the temperature during treatment with sodium periodate
Activity of biomass, mmol/g wet cells per h
Culture temperature during the treatment, ~
E. aroidea
B. subtilis
A. faecalis
Pseudomonas sp. Brevibacterium sp.
Before treatment
1.0 2.1 0.8 2.4 9.3
1.1 2.1 0.8 2.6
10.0
2.0 4.4 2.8
19.7 20.5
5 7
3.0 1.7 6.0 3.7 3.7 3.5
25.5 20.5 28.7 18.3
10
0.9 1.2 2.6
16.8 11.1
15
0.3 u
0.4 8.8 6.4
Table 5. Dependence of the cell activity on the concentration of PDAI (treatment for 20 h)
Activity, mmol/g cells per h
Culture
E. aroidea
B. subtilis
A. faecalis
Pseudomonas sp. Brevibacterium sp.
pH
8.5 8.5 7.5 7.5 7.0
3.0 6.0 3.7
25.5 28.7
0.05
�9 6.5 9.7 4.1
26.3 33.3
concentration of PDAI (0.5 ml), %
0.10
7.8 15.9 5.0
28.7 56.5
0.15
7.6 10.1 4.7
28.7 50.1
0.25
7.5 8.3 4.0
26.7 29.9
0.50
7.0 7.7 3.8
25.0 22.7
1.0
7.0 5.4 3.6
23.0 16.1
2.0
2.5
2.9 19.0 10.1
Table 6. Dependence of the cell activity on the volume of 0.1% PDAI (treatment for 20 h)
Activity, mmol/g cells per h
Culture
E. aroidea
B. subtilis
A. faecalis
Pseudomonas sp. Brevibacterium sp.
pH
0
8 . 5 3 . 0
8.5 6.0 7.5 3.5 7.5 25.5 7.0 28.7
volume of PDAI, ml
0.10
6.1 9.7 4.6
28.5 41.5
0.25
8.3 15.9 5.5
30.0 56.4
0.50
7.8 13.8 5.0
28.7 50.0
1.0
7.8 8.8 3.8
25.0 31.6
2.0
7.5 4.5 3.7
23.8 25.4
3.0
4.2
3.2 21.3 22.1
activities of producers of aspartase, L-aspartate-13- decarboxylase, and fumarase, were 8.5, 7.5, and 7.0, respectively. For aspartase, fumarase, and L-aspartate- ~-decarboxylase from Pseudomonas sp., pH can range from 7.0 to 10.0; and for L-aspartate-13-decarboxylase from A. faecalis, from 7.0 to 8.5 (Table 7). The optimal time of cell treatment was 20 h, but 10-25 h was also allowable (Table 8).
Thus, the following best conditions for the cell link- ing were established: 0.25 ml of 0.1% PDAI solution per g wet cells at pH 7.0-8.5 for 20 h at room tempera- ture. This treatment resulted in a gel of immobilized cells of high enzymatic activity, which was higher than the activity of initial cells.
In addition to PDAI, other diamines and dihy- drazides were also studied, such as N,N'-n-diphenylene
diasparimide (DPDAI), dihydrazides of 2-(2'-oxyhep- tyl) and 2-(2'-dioxyoctyl) succinic acid (DOHS and DOOS, respectively), and also dihydrazides of 2-(2'- oxyheptyl)- and 2-(2'-oxyoctyl)-glutaric acid (DOHG and DOOG, respectively), but they were found to be less efficient (Table 9). The data indicate that the pro- posed approach is very promising. Thus, this method allowed a 7.5- to 10-fold increase in the activity of immobilized cells. However, the resulting gel was insufficiently compact and not convenient for use in flow systems. To overcome this disadvantage, the cells, after treatment with sodium pedodate and PDAI, were additionally linked with glutaraldehyde, transferred to a vacuum-drier drum, and dehydrated at 40-45~ As a result, soft gel pieces were changed to a solid stable material. The resulting dry particles of the product were
APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 36 No. 3 2000
A NEW METHOD FOR IMMOBILIZATION OF MICROBIAL CELLS 313
Table 7. Dependence of the activity of microbial cells on pH of PDAI solution (0.25 ml of 0.1% PDAI solution per g cells, 20 h)
Activity of the biomass, mmol/g wet cells per h
Culture pH of PDAI solution
E. aroidea B. subtilis
A. faecalis Pseudomonas sp. Brevibacterium sp.
After treatment with sodium periodate
3.0 6.0
3.7 25.5 28.7
7.0
3.5 6.0 4.7
26.0 56.5
7.5
4.0 8.3
5.5 30.0 54.9
8.0
5.1 10.1
5.5 27.9 50.1
8.5
8.3 15.9 4.5
26.8 49.6
9.0
7.3 15.0
3.1 25.5 41.1
9.5
5.5 13.2
2.7 25.0
38.4
10.0
3.5
4.3 2.5
25.0 33.0
Table 8. Dependence of the cell activity on the time of treatment with 0.25 ml of 1% PDAI
Activity, mmol/g cells per h
Culture
E. aroidea B. subtilis A. faecalis Pseudomonas sp.
Brevibacterium sp.
pH
8.5 8.5 7.5 7.5
7.0
3.0 6.0 3.7
25.5 28.7
10
4.0 8.5 3.8
27.5
30.1
time of the treatment, h
15 20
6.2 8.3 12.1 15.9 4.3 5.5
29.2 30.0
46.9 56.5
25
7.8 11.8
5.0 28.9
55.1
30
7.3 10.6 4.7
25.1
50.6
Table 9. Effects of diamines on the activity of cells during their immobilization*
Activity, mmol/g cells per h
Culture E. aroidea A. faecalis Pseudomonas sp.
In Im In Im In Im
DPDAI
DOHS
DOOS DOHG DOOG
1.0
1.0 1.0
1.0 1.0
6.4
7.1 7.9 6.9 7.8
0.7 0.7
0.7
0,7 0.7
4.5
3.7
5.0 4.5 3.8
2.1
2.1
2.1 2.1 2.1
6.4 11.1
14.5
9.8 13.3
* Activity of the cells: In, initial (intact); Im, immobilized.
characterized by sufficient stability and structural strength and high enzymatic activity, which was inher- ent in microbial cells. The catalyst was suitable for repeated use in reactors of periodic operation or in col- umn reactors.
The best results were obtained with the use of 0.15 weight parts of glutaraldehyde per 1.0 weight part of concentrated cells of E. aroidea, B. subtilis, and Brevibacterium sp. and with 0.10 weight parts of glut- araldehyde for concentrated cells of Pseudomonas sp. and A. faecalis (Table 10).
The resulting biocataIyst is especially valuable when high-molecular-weight compounds are used as substrates. A purified enzyme whose isolation is an
expensive procedure or cells immobilized by incorpo- ration or adsorption are usually employed in these pro- cesses. However, in the former case, diffusional limita- tions prevent the complete conversion of the polymer, and the rate of substrate flow through the biocatalyst layer is too low. In the latter case, the cells are rapidly washed out of the column. Almost none of the biocata- lyst obtained by the method described above was washed out of the column. This method allows us to link cells of different microorganisms and thus to pro- duce multienzyme catalysts.
The enzyme stability is known to increase after their intramolecular linking by various bifunctional agents
APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 36 No. 3 2000
314 ABELYAN
Table 10. Effect of glutaraldehyde on the activity of biocatalysts
Glutaraldehyde, Activity, % of the maximum
weight parts E. aroidea B. subtilis Pseudomonas sp. A. faecalis Brevibacterium sp.
0.06 0.08 0.10 0.15 0.20 0.40
83 88 92
100 89 89
71 91 98
100 91 90
83 94
100 93 88 87
83 96
100 90 81 76
85 90 95
100 90 89
due to an increased conformational rigidity of the pro- tein.
A similar increase in stability was also observed in cells treated with sodium periodate and PDAI. Thus, E. aroidea and B. subtilis cells, which were linked in this manner and incorporated in polyacrylamide gel (PAAG), lost only 7-8% of their initial activities over 90 days of uninterrupted operation, whereas the half- life of nonlinked cells under the same conditions was only 30 days (figure).
This effect was likely due to binding of free alde- hyde groups on the cell wall surface to free amino groups of PAAG during polymerization. The prelimi- narily stabilized cell aggregates were attached to the gel matrix by multiple covalent bonds and thus became a structural part of the gel matrix as though they were a monomer during the polymerization.
Thus, a new carder-free method of microbial cell immobilization has been designed. This method is especially valuable for production of IC of extreme-
A,% 100
80
60
40
20
1
2
3
10 30 50 I I
70 90 Day
Stability of IC with an aspartase activity (a solution of ammonium fumarate (1 M, pH 8.5) was passed through a column (2 x 20 cm) with the biocatalyst at the specific velocity of 0.4 h -1 at 37~ (1) Linked cells of E. aroidea, (2) linked cells of B. subtilis, (3) nonlinked cells of E. aroi- dea, (4) nonlinked cells of B. subtilis, A, activity, % of the maximum.
philic microorganisms and is promising for cases when high-molecular-weight compounds are used as sub- strates. The resulting biocatalysts were characterized by high activity and stability. This is a new approach to the production of multienzyme catalysts based on microbial cells.
ACKNOWLEDGMENTS
I am grateful to V.S. Meliksetyan, A.P. Antonyan, and S.N. Bagdasaryan (Institute of Microbiology, National Academy of Sciences of Armenia) for their help in this work.
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APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 36 No. 3 2000