ISSN 0003-6838, Applied Biochemistry and Microbiology, 2007, Vol. 43, No. 2, pp. 173–177. © Pleiades Publishing, Inc., 2007. Original Russian Text © A.Yu. Maksimov, Yu.G. Maksimova, M.V. Kuznetsova, V.F. Olontsev, V.A. Demakov, 2007, published in Prikladnaya Biokhimiya i Mikrobiologiya, 2007, Vol. 43, No. 2, pp. 193–198.
Immobilization of Rhodococcus ruber Strain gt1, Possessing Nitrile Hydratase Activity, on Carbon Supports A. Yu. Maksimova, Yu. G. Maksimovaa, M. V. Kuznetsovaa, V. F. Olontsevb, and V. A. Demakova a
Institute of Ecology and Genetics of Microorganisms, Uralian Division of the Russian Academy of Sciences, Perm, 614081 Russia e-mail:
[email protected] b OAO Sorbent, Perm, 614113 Russia Received December 27, 2005
Abstract—Rhodococcus ruber strain gt1, possessing nitrile hydratase activity, was immobilized by adsorption on carbon supports differing in structure and porosity. The adsorption capacity of the supports towards cells, the substrate of the nitrile hydratase reaction (acrylonitrile), and the product (acrylamide) was studied. Also, the effect of immobilization on nitrile hydratase activity of bacteria was investigated, and the operational stability of the immobilized biocatalyst was determined. It was shown that crushed and granulated active coals were more appropriate for immobilization than fibrous carbon adsorbents. DOI: 10.1134/S000368380702007X
Bacteria possessing nitrile hydratase activity and able to convert nitriles to corresponding amides are used in the biocatalytical acrylamide industry [1]. The advantages of these biotechnological processes over chemical ones are the following: mild reaction conditions, high specificity, and complete conversion of the substrate [2]. The efficiency of industrial biotechnological processes can be improved by immobilization of biocatalyst cells. This immobilization provides advantages over both free cell cultures and immobilized enzymes owing to more suitable conditions of biocatalytic processes [3–5]. Adsorption on the surface of a solid support is one of the simplest and readily available methods of microbial cell immobilization. Promising adsorbents for immobilization include activated carbons and other carbon materials, which demonstrate high chemical and biological stability, mechanical strength, permeability for substrates, large specific surface, and the possibility of obtaining technologically appropriate forms (granules, fabrics, fibers, or modules). The arrangement of pores in active carbons, formed by interstices between individual graphite-like crystals, and the presence of free valences in carbon atoms on crystal surfaces support their ability to bind, chemically and sorptionally, various substances [6, 7]. However, little attention is currently paid to using carbon materials for cell immobilization. This study is dedicated to the effect of adsorptional immobilization of Rhodococcus ruber strain gt1 cells, possessing nitrile hydratase activity, on the efficiency of bioconversion of acrylonitrile to acrylamide.
MATERIALS AND METHODS Immobilization was performed with Rhodococcus ruber strain gt1 cells, isolated during previous selection, which possessed nitrile hydratase activity [8]. The following carbon adsorbents were used as supports (see table): crushed activated birch charcoal BAU, granulated activated carbon FTD, activated carbon fabric Ural, activated nonwoven fabric Voilok (Felt), activated carbon material FAS, raw charcoal crushed to powder with 50–300 µm particles (Russia), and crushed activated carbon NORIT PK 1–3 (Netherlands). The R. ruber gt1 cells were grown on synthetic medium N of the following composition, g/l: glucose, 1.0; NH4Cl, 0.3; KH2PO4 , 1.0; K2HPO4 · 3H2O, 1.6; NaCl, 0.5; MgSO4 · 7H2O, 0.5; CaCl2 , 0.005; FeSO4 · 7H2O, 0.01; and CoCl2 · 6H2O, 0.01; pH 7.4 [8]. Bacteria were immobilized by incubation of adsorbents with cells grown to the stationary phase for 35 min with shaking at 100 rpm. The adsorbents were collected by filtration through a “blue tape” paper filter and washed with 10 mM potassium phosphate buffer. The filtrate was refiltered. The amount of adsorbed cells was determined from the difference between the optical densities (D540) of the culture liquid before and after adsorption. The adsorption capacity with respect to cells was expressed in mg of dry cell weight per one gram of the adsorbent. Acrylonitrile conversion with immobilized R. ruber gt1 biomass was performed for 10 min in 0.01 M potassium phosphate buffer pH 7.5. The reaction was terminated by the addition of HCl to the final concentration 2%. Acrylamide concentration was determined from
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Carbon supports Trademark
Pore structure parameters* Bulk density, 3 g/dm VΣ, cm3/g Vmi, cm3/g Vme, cm3/g Vma, cm3/g dma, µm
Feedstock
Activated birch charcoal BAU-A
Birch charcoal
220 ± 20
1.65–1.80 0.22–0.25
0.08–0.10
1.35–1.45
5–40
FTD
Phenol–formaldehyde resin
480 ± 20
0.80–0.85 0.50–0.60
0.12–0.14
0.08–0.10
7–15
Active fabric Ural
Viscose fabric
200 ± 30** 0.75–0.85 0.32–0.35
0.13–0.15
0.30–0.35
5–10
Active nonwoven fabric Voilok
Viscose–Voilok
250 ± 20** 0.60–0.68 0.25–0.31
0.08–0.15
0.35–0.36
–
FAS
Furyl resin
480 ± 20
0.82–0.86 0.48–0.50
0.20–0.30
0.14–0.16
–
NORIT PK 1–3
Peat
530 ± 20
0.80–0.85 0.38–0.43
0.06–0.07
0.24–0.28
2–30
Raw birch charcoal
Birch wood
280 ± 5
1.00–1.20
0.02–0.05
0.95–1.15
5–40
0
* VΣ, total pore volume; Vmi, micropore volume; Vme, mesopore volume; Vma, macropore volume; dma, macropore diameter; “–”, not determined. ** Surface density, g/m2.
optical density (D260), measured spectrometrically on an SF-46 spectrometer (LOMO, Russia). The nitrile hydratase activity of R. ruber gt1 cells was estimated from the change of acrylamide concentration in the reaction mixture after 10 min of the reaction. The specific activity of the enzyme was expressed in micromoles of acrylamide produced for 1 min per 1 mg of dry cell weight (µmol/mg min) [1]. The adsorption capacity of the supports with respect to acrylonitrile and acrylamide was estimated from the difference between the concentration of dissolved substance before and after adsorption. Acrylonitrile concentration was measured with a Chrom-5d chromatograph (LP, Czech Republic) with flame-ionization detection and a 2 m long steel column with Polysorb-1 (Reakhim, Russia), fraction 0.25–0.5 mm. Solution of pure acrylonitrile was used as a reference [8]. mg/g 16 14 12 10 8 6 4 2 0
1
2
3
4
5
6
7
Fig. 1. Adsorption capacity of supports towards R. ruber gt1 cells (mg/g): 1, BAU; 2, NORIT PK 1–3; 3, FTD; 4, raw charcoal; 5, Ural; 6, Voilok; 7, FAS.
The operational stability of immobilized and free cells was estimated from the preservation of nitrile hydratase activity during a series of batch conversions of acrylonitrile added to 10% in 10 ml of 0.01 M potassium phosphate buffer pH 7.5 in each cycle. The full conversion cycle was carried out for 20 min, and the adsorbents were washed with potassium phosphate buffer. The reaction was catalyzed by cells, either free (2.5 mg of dry weight) or immobilized, on carbon supports. For investigation of bacterium adsorption during bacterial growth, R. ruber gt1 cells were grown with adsorbents in Erlenmeyer flasks with synthetic medium N, containing 5 mM ammonium sulfate and 0.1% glucose at constant shaking at 150 rpm for 168 h. RESULTS AND DISCUSSION Adsorption capacity of supports towards cells. As shown in Fig. 1, high adsorption capacity, up to 13– 15 mg dry cells/g support was demonstrated by activated crushed coals BAU, FTD, NORIT PK 1–3, and raw charcoal. Fibrous materials (Voilok, Ural, and FAS) had lower adsorption capacities: 7.5, 9.5, and 8 mg/g adsorbent, respectively. The maximum amount of cells was adsorbed by the activated woven fabric Ural as late as one day after incubation, whereas the maximum adsorption on other supports was achieved within 30 min. It is known that cell adsorption depends on the accessible surface area, which is formed mainly by macropores, larger than the microbial cell [5, 6]. Activated carbon BAU has a large macropore volume: 1.35–1.45 cm3/g, or 80–82% of the total pore volume (table). The diameters of macropores in the supports under study are within 2–40 µm, which allows efficient adsorption of the cells, which are 1–7 µm in size. The
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granulometry of carbon supports is also important for cell adsorption. The fine-dispersed state of FTD and raw charcoal allows binding of rhodococcal cells close to that by BAU. Of the supports studied, Voilok has the least total pore volume, 0.60–0.68 cm3/g, and, in FAS and Ural, micro- and mesopores add up to a volume larger than the volume of macropores, which affects cell adsorption. Adsorption capacity of supports towards acrylamide, the product of the enzymatic reaction. An important stage of biocatalytic synthesis is separation of the reaction product from the biocatalyst. In processes with immobilized catalytically active cells, binding of the product to the support should be regarded. The adsorption capacity of carbon supports towards acrylamide is shown in Fig. 2. Supports with immobilized cells adsorbed less acrylamide than cell-free ones. The largest amount of acrylamide was adsorbed by activated carbon fabric Ural: 27 mg/g support without cells and 17 mg/g with cells. Raw charcoal adsorbed virtually no acrylamide. The difference between the adsorption of the reaction product by a free support and the support loaded with cells was well-pronounced in the case of crushed active carbon BAU (22 and 7 mg/g support, respectively). The decrease in acrylamide adsorption on supports after cell immobilization is likely to be related to the fact that immobilized cells hamper access to part of the micropores. An amount of acrylamide can also be adsorbed on the surface of free macropores. The total volume of raw charcoal pores is constituted mainly by macropores, which explains the poor adsorption of acrylamide molecules and its complete absence after cell immobilization. In contrast, active Ural fabric retains the ability to adsorb much acrylamide even after cell immobilization, probably, because the total surface of Ural is equally contributed by micro- and macropores, which form interfiber space rather than a network structure, as in granulated coals [5]. Adsorption capacity of supports towards acrylonitrile, the substrate of the enzymatic reaction. Adsorption was performed in 10 ml of 10% aqueous acrylonitrile. Carbon supports were added in 1 g weights and incubated for 30 min. As evident from Fig. 3, BAU, Voilok, and FAS had high adsorption capacities, up to 350–400 mg/g. The lowest adsorption was observed on raw charcoal. Acrylamide was also detected during incubation of R. ruber gt1 cells with acrylonitrile adsorbed on supports but absent from the medium, probably owing to its desorption. Effect of the substrate concentration on the rate of the nitrile hydratase reaction performed by immobilized cells. The dependence of the rate of the nitrile hydratase reaction in the suspension of R. ruber gt1 cells on acrylonitrile concentration is shown in Fig. 4. The nitrile hydratase activity linearly increased APPLIED BIOCHEMISTRY AND MICROBIOLOGY
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mg/g 30
I
25
II
20 15 10 5 0
1
2
3
4
5
6
7
Fig. 2. Adsorption capacity of supports towards acrylamide (mg/g): I, acrylamide adsorption by cell-free supports; II, acrylamide adsorption by supports with immobilized cells. Designations follow Fig. 1.
g/g 0.5 0.4 0.3 0.2 0.1 0
1
2
3
4
5
6
7
Fig. 3. Adsorption of acrylonitrile, the substrate of the nitrile hydratase reaction, by carbon supports (g/g). Designations follow Fig. 1.
µmol/mg·min 600 400 200
0
0.2
0.4
0.6
0.8
1.0
1.2 å
Fig. 4. Acrylonitrile concentration dependence of the nitrile hydratase activity (µmol/mg min) of R. ruber gt1 cells in suspension.
with acrylonitrile concentration (from 2 to 50 mM). Further increase in the substrate concentration did not increase the reaction rate substantially.
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% 100 80 60 40
4
3
2
20
5
1 0
1
2
3
4
5
6
7
8
9
6
7 10 1
2
3
4
5
6
7
8
9
10 Cycle
Fig. 5. Operational stability of the immobilized biocatalyst (% of the starting activity), containing 2.5 mg of dry cell weight and possessing the starting activity 200 µmol/mg min in the sequence of 20-min cycles of conversion of 10% acrylonitrile (10 ml). Designations follow Fig. 1.
We carried out the nitrile hydratase reaction with acrylonitrile as the substrate and the adsorbed biocatalyst. It was found that the reaction rate corresponding to the highest activity of intact cells was achieved at substrate concentrations within 0.8–1.2 M with rhodococcal cells immobilized on carbon supports. The higher substrate concentration required for the reaction with immobilized cells is probably related to substrate adsorption on the support. The immobilization by adsorption does not cause loss of the enzymatic activity. Operational stability of cells immobilized on carbon supports in sequential reaction cycles. Sequential cycles of acrylonitrile batch conversion showed that the activity of cells immobilized on fibrous carbon supports began to decrease after 5 cycles (Fig. 5). The biocatalyst immobilized on BAU, FTD, or FAS retained its activity for 6 cycles, and on raw charcoal or NORIT
PK 1–3, for 7 cycles. The nitrile hydratase activity of suspended cells with complete conversion of 10% acrylonitrile decreased twofold in the second cycle and tenfold by the sixth cycle with reference to the starting activity (Fig. 6). Thus, immobilization by adsorption stabilizes the nitrile hydratase activity in spite of high concentrations of the toxic substrate in the solution. Growth of R. ruber gt1 cells with the presence of supports. We studied the effect of adsorptional immobilization on R. ruber gt1 cells grown with the presence of carbon adsorbents. Adsorption on carbon supports had no adverse effect on cell viability. The amount of cells grown on adsorbents was larger than in suspension, as judged from the increase in total activity. The yield of acrylamide in the nitrile hydratase reaction with cells adsorbed during 7-day growth was determined (Fig. 7). The production of acrylamide by cells % 160
% 100
120
80 60
80
40
40
20 0
0 1
2
3
4
5
6
7 Cycle
Fig. 6. Operational stability of R. ruber gt1 cells (% of the starting activity) in suspension (2.5 mg of dry cell weight) with the starting activity 200 µmol/mg min in the sequence of 20-min cycles of conversion of 10% acrylonitrile (10 ml).
1
2
3
4
5
6
7
Fig. 7. Production of acrylamide (% of the amount of acrylamide produced by suspended cells in 10-min conversion of 10% acrylonitrile (10 ml)) by R. ruber gt1 cells adsorbed on supports during 7-day growth. Production of acrylamide by the 7-day suspension of R. ruber gt1 cells (27 mg of dry cell weight) is taken to be 100%. Designations follow Fig. 1.
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adsorbed on active crushed coals (BAU or NORIT PK 1-3) or raw charcoal exceeded that in the control R.ruber gt1 suspension grown for 7 days without adsorbents by 20–50%. Cells adsorbed on active fabric Ural or FTD produced the same amounts of acrylamide as free ones, and the production on Voilok was 70% of the control. The least acrylamide production was observed with cells adsorbed during growth on FAS: only 3.5% of the control value. Our results indicate that application of active crushed or granulated coals as supports for immobilization of the strain R. ruber gt1 is preferable over fibrous carbon materials or FAS. ACKNOWLEDGMENTS This study was supported by the Program of Cooperative Studies of the Ural Division of the RAS with scientists of the Siberian Division of the RAS and by the Russian Foundation for Basic Research, project no. 04-04-97514. REFERENCES 1. Astaurova, O.B., Leonova, T.E., Polyakova, I.N., et al., Prikl. Biokhim. Mikrobiol., 2000, vol. 36, no. 1, pp. 21–25.
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2. Zabaznaya, E.B., Kozulin, S.V., and Voronin, S.P., Prikl. Biokhim. Mikrobiol., 1998, vol. 34, no. 4, pp. 377–381. 3. Immobilized Cells and Enzymes-a Practical Approach, Woodward, J., Ed., Washington, DC: IRL, 1985. Translated under the title Immobilizovannye kletki i fermenty. Metody Moscow: Mir, 1988. 4. Halgas, J., Biocatalysts in organic synthesis, Amsterdam: Elsevier, 1988. Translated under the title Biokatalizatory v organicheskom sinteze, Moscow: Mir, 1991. 5. Sinitsyn, A.P., Rainina, E.I., Lozinskii, V.I., and Spasov, S.D., Immobilizovannye kletki mikroorganizmov (Immobilized Cells of Microorganisms), Moscow: Mosk. Gos. Univ., 1994. 6. Davidenko, T.I., Ugol’nye materially—nositeli dlya immobilizatsii fermentov i kletok (Carbon Materials As Carriers for Immobilization of Enzymes and Cells), Odessa: FKhI NAN Ukraine, 1995. 7. Olontsev, V.F. and Bezrukov, R.A., Rossiiskie aktivnye ugli (Russian Active Carbons), Moscow: GU VShE, 1999. 8. Maksimov, A.Yu., Kuznetsova, M.V., Ovechkina, G.V., et al., Prikl. Biokhim. Mikrobiol., 2003, vol. 39, no. 1, pp. 63–68.
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