The use of transition metal-based supports for cell immobilization has proved its feasibility in some fermentative or bioconversion processes with potential for commercial-scale application in the food industry. It was therefore logical to evaluate the validity of this immobilization method within the development of other relevant bioprocesses, such as steroid bioconversions. Among the transition metal-based supports, titanium-based supports are by far the most widely used for whole cell immobilization. In order to illustrate this concept, some practical approaches for the immobilization of yeast and bacterial cells onto titanium-based supports, aiming for the production of biocatalysts with application in bioconversion processes, are presented in this work
Key Words: Immobilization; titanium; transition metals; whole cells. 1. Introduction
Among the multiple methodologies used for the immobilization of whole cells, an approach based on transition metal chemistry has proved feasible (1-5), particularly in processes related to the food industry, namely in wine-making and brewing (6-9). Other examples include bioconversion of sterols (5) and steroids (10) or vinegar production (11). This methodology is based on the nontoxicity of transition metal oxides toward microbial cells (12), and its application has also been extended to the immobilization of whole cells onto different inorganic or organic supports (e.g., celite, DEAE-cellulose) treated with a given transition metal. Although oxides of iron (III), vanadium (III), tin (IV), zirconium (IV), and titanium (IV) have been used for the immobilization of biomolecules (1,13,14), the latter is clearly the most commonly used transition metal for whole cell immobilization. Using a gelatinous hydrous metal oxide, cells can be immobilized onto transition metal supports. This approach involves the replacement of hydroxyl groups in the surface of the metal hydroxide with ligands from the cell, leading to partial covalent bonds. The preparation of hydrous titanium (IV) oxide is made from the corresponding tetrachloride derivative, which is neutralized to pH 7.0 by addition of ammonium hydroxide. The excess ammonium ions are washed with a physiological saline solution from the resulting metal hydroxide, which can then be used as immobilization support. Once the hydrous titanium oxide is added to a cell suspension, cells tend to aggregate and a precipitate is rapidly obtained, which can easily be recovered by centrifugation (15). This straightforward and easy to follow procedure is hampered however by cell loss and metal leakage (15). A different cell immobilization procedure involving transition metal chemistry, based on cell adsorption to the support, has also been reported (4,5,10). Adsorption is a simple and cheap method of cell immobilization, and therefore promising, particularly if a commercial scale process is envisaged. The cell suspension can contact the support in either a stirred tank or can be pumped through a bed of support, to promote cell adsorption. Even the risk of desorption can be turned into an advantage, because the matrix can be easily regenerated, by allowing the cells to desorb, once their catalytic activity decreases (15).
The support used for cell immobilization can be based on pure titanium (IV) oxide in powder, granular, or honeycomb monolith structure (4,5). A more conventional support activated with a transition metal salt can also be used (10,16).
2.1. Immobilization of Yeast Cells on Hydrous Titanium Oxide (1,16,17)
1. Titanium (IV) chloride 150 g/L solution in 150 g/L Hcl (see Note 1).
2. Ammonium hydroxide 2.0 M solution (handle carefully: irritant)
3. Saccharomyces cerevisiae cells
4. 0.9% (w/v) Physiological NaCl solution.
5. Fume hood and disposable gloves.
2.2. Immobilization of Arthrobacter simplex Cells on Titanium-Activated Inorganic Supports
1. 42 g/L Potato dextrose agar slants.
2. Sterile solution of 10 g/L yeast extract either with or without 0.3 g/L cortisol in 20 mM potassium phosphate buffer, pH 7.0.
3. Celite 80-120 mesh.
4. Titanium (IV) chloride 150 g/L solution in 100 g/L HCl (handle carefully: highly corrosive).
5. 20 mM Potassium phosphate buffer, pH 7.0.
2.3. Immobilization of Mycobacterium spp. Cells Onto Titanium Oxide
1. 42 g/L Potato dextrose agar slants.
2. Sterile solution of 10 g/L yeast extract in 20 mMpotassium phosphate buffer, pH 7.0.
3. Sterile solution composed of 10 g/L fructose, 2 g/L ammonium chloride, 0.1 g/L magnesium sulfate, 0.025 g/L calcium chloride, 0.78 g/L Tween-20, and 0.1 g/L sitosterol in 20 mMpotassium phosphate buffer, pH 7.0.
4. Granular or powdered TiO2.
5. 20 mM Potassium phosphate buffer, pH 7.0.
6. Mycobacterium spp. NRRL B-3805 cells.
3.1. Immobilization of Yeast Cells on Hydrous Titanium Oxide
1. Slowly add a 2 Mammonium chloride solution to 10 mL of a 150 g/L solution of titanium (IV) chloride until neutrality, pH 7.0 (see Note 2).
2. Wash the precipitate with 3 x 25 mL of a 0.9% (w/v) NaCl solution. The hydrous titanium oxide is ready to be used as cell support.
3. Add 10 mL of a 2% (w/v) of a yeast cell suspension in 0.9% (w/v) NaCl solution to the support prepared in step 2. Stir gently (e.g., 120 rpm in an orbital shaker) at room temperature for 5 min to allow cell immobilization.
4. Allow the mixture to settle down at room temperature, until a clear supernatant is observed.
5. Centrifuge at low speed and discard the supernatant. Determine hydrolytic activity if the immobilized biocatalyst as described in Subheading 3.2.
3.2. Determination of the Activity of Yeast Cells Immobilized on Hydrous Titanium Oxide
Activity determination is based on the invertase enzymatic activity of the immobilized cells. This is measured using as substrate a 2.0% (w/v) solution of sucrose in 20 mM sodium acetate buffer, pH 4.5.
1. Add 100 mg of immobilized biocatalyst to 5 mL of a 2.0% (w/v) solution of sucrose in 20 mM sodium acetate buffer 20 mM, pH 4.5, at 45°C.
2. At zero time and at various time intervals along an incubation period of 30 min at 45°C, take 100 |L aliquots and place the samples in an ice bath to stop the reaction.
3. Thaw the samples to room temperature, and add 100 |L of DNS reagent. Mix thoroughly and heat in a boiling water bath for 5 min. Cool to room temperature, add 1 mL of distilled water and measure the absorbance of the solutions at 540 nm. The concentration of the reducing sugars formed may be assessed by using a calibration curve determined according to Note 3.
3.3. Immobilization of A. simplex Cells on Titanium-Activated Inorganic Supports (10)
1. Cell growth and harvest: A. simplex (ATCC 6946) can be grown in 1-L shake flasks containing 250 mL of growth medium, with 150 rpm orbital shaking at 30°C.
2. The growth medium contains 10 g/L of yeast extract in 20 mMphosphate buffer, pH 7.0. The inoculum is a 25-mL portion of a 17-h culture on the same medium, grown in the described conditions.
3. Cortisol (inducer of the steroid A1-dehydrogenase) powder is added to the fermentation medium 8 h after inoculation to a final concentration of 0.3 g/L.
4. Cells can be harvested 18 h after induction by centrifugation at 9000 rpm and 10°C and washed twice with a 2% (v/v) methanol solution in 20 mM phosphate buffer, pH 7.0.
5. The wet cell paste, containing 220 of cell dry weight/g (determined at 80°C) can be stored at -20°C.
6. Thoroughly wash Celite with distilled water and acetone and dry the support at 80°C overnight.
7. Add 6.5 mL of a 150 g/L solution of titanium chloride in 150 g/L HCl to 1 g of Celite and heat up to 80°C under reflux for about 2.5 h. A hydrous titanium oxide layer is formed that adheres to Celite.
8. Wash thoroughly the support with distilled water and phosphate buffer and filter off with qualitative filter paper.
9. Add 20 mL of a cell suspension, roughly 0.3% (w/v), based on dry cell weight, in 20 mM phosphate buffer, pH 7.0, to 1 g of the activated support and stir the mixture for 5 to 7 h, at 30°C and 150 rpm, in an orbital shaker.
10. Periodically monitor the optical density (640 nm) of the supernatant. The cell-loaded support is then filtered off with qualitative filter paper, thoroughly washed with phosphate buffer and used for bioconversion.
3.4. Determination of the Activity of A. simplex Cells Immobilized on Titanium-Activated Inorganic Supports (10,18)
The determination of biocatalytic activity is based on the A1-dehydrogenation of cortisol to prednisolone.
1. Add 500 mg of immobilized biocatalyst to 5 mL of a 1.0 g/L solution of cortisol in ^-decanol (previously saturated with 20 mM phosphate buffer, pH 7.0), containing 80 mg/L of menadione (external electron acceptor; see Note 4).
2. Collect 100 |L samples at time zero and along the time course of the byconversion up to 24 h and dilute the samples with chloroform containing 120 mg/L cortisone (internal standard) and evaluate the steroid content by HPLC.
3. Steroid analysis is performed by high performance liquid chromatography (HPLC) using a Lichrosorb Si-60 column 250 x 4 mm; 10 |im particle diameter (VWR), with a mixture of (v/v) dichloromethane (94%) methanol (3%) and glacial acetic acid (3%) as mobile phase, at a flow rate of 1.0 mL/min. The products are detected at 254 nm and matched to pure cortisol and prednisolone.
3.5. Immobilization of Mycobacterium spp. Cells Onto Titanium Oxide (5)
The determination of biocatalytic activity is based on the side-chain cleavage of sitosterol to 4-androstene-3,17-dione.
1. Titanium oxide must be thoroughly washed with water and acetone and dried overnight at 80°C
2. Cell growth and harvest: cells can be grown in 250-mL Erlenmeyer flasks containing 50 mL of a medium composed of: 10 g/L fructose, 2 g/L ammonium chloride, 0.1 g/L magnesium sulfate, 0.025 g/L calcium chloride, 0.8 g/L Tween-20, and 0.1 g/L sitosterol in 20 mMpotassium phosphate buffer, pH 7.0. Growth is carried out in an orbital shaker at 30°C and 200 rpm. The inoculum is a 0.5-mL portion of a 17-h culture on yeast extract (10 g/L) in 20 mM potassium phosphate buffer, pH 7.0, grown in the described conditions. Cell adsorption is promoted by adding 2 g of support to the cell culture when an optical density (640 nm) is achieved. Growth is allowed to proceed for a further 4-h period, allowing cell immobilization to occur. The cell loaded support is then harvested by filtration on qualitative filter paper, thoroughly washed with 20 mMphosphate buffer, pH 7.0, and used for bioconversion trials.
3. Dry samples of the cell-loaded support and assay for protein content according to Lowry and co-workers (18), following cell hydrolysis by heating at 100°C for 20 min in a 1 M solution of NaOH (19).
3.6. Determination of the Activity of Mycobacterium spp. Cells
Immobilized on Titanium Oxide (5)
1. Add 500 mg of support with immobilized biocatalyst to 5 mL of a 1 g/L suspension of sitosterol in 20 mM phosphate buffer, pH 7.0, and incubate in screw-capped bottles placed in an orbital shaker at 200 rpm and 30°C.
2. Collect 100 |L samples at time zero and along the time course of the byconversion up to 72 h, extract the samples with 400 |L of n-heptane containing 200 mg/ L of progesterone (internal standard) and evaluate the steroid content by HPLC.
3. Steroid analysis was performed by HPLC using a Lichrosorb Si-60 column 250 x 4 mm; 10 |im particle diameter (VWR), with n-heptane containing 5% (v/v) etha-nol as the mobile phase at a flow rate of 1.0 mL/min. The products were detected at 215 nm and matched to pure sitosterol and androstenedione.
1. Handle carefully: highly corrosive.
3. To 100 |L aliquots of D-glucose standard solution in a 0 to 1000 mg/L in 20 mM sodium acetate buffer, pH 4.5, add 100 ||L of DNS reagent, mix thoroughly and heat in a boiling water bath for 5 min. Cool to room temperature, add 1 mL of distilled water and measure the absorbance of the solutions at 570 nm.
4. Incubate in screw capped bottles placed in an orbital shaker at 200 rpm and 30°C.
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