evaluated the performance of fluidized bed reactor consisting of immobilized Sac-charomyces cerevisiae and amyloglucosidase for bioconversion of soluble starch and yeast extracts. The biocatalyst exhibited no significant loss of activity during many weeks of continuous operation. Recently, Krishnan et al. (8) have looked at the economics of fuel ethanol production from cornstarch. The glucose fermentation step was carried out in a fluidized bed reactor using Zymomonas mobilis entrapped in K-carrageenan beads. It was estimated that an operating cost saving in the range of 1.1-1.3 cents/gal was possible with fluidized bed reactor technology for a 15-million gal/yr ethanol plant.
In another approach, it has been found that if the bed consists of magnetically susceptible particles, application of a magnetic field facilitates control of fluidiza-tion. In such magnetically stabilized fluidized beds, a weak stable external magnetic field is applied axially, relative to the flow of the fluidizing medium. This makes it possible to operate at higher fluidization velocities. Webb et al. (9) have provided a good discussion on the advantages of such beds with a focus on improved mass transfer studied with a number of immobilized enzyme systems.
The protocol described in this chapter illustrates a somewhat novel concept in which preparations of two different enzymes, each immobilized on the same matrix, were premixed to carry out a sequential conversion in the fluidized bed reactor.
2.1. Assay of Pullulanase and Glucoamylase
1. Pullulanase from Bacillus acidopullulyticus (supplied as Promozyme, Novozymes®, Krogshoejvej, Denmark).
2. Glucoamylase from Aspergillus niger (supplied as Palkodex, Maps India Ltd., Ahmedabad, India).
3. Soluble starch from potato (E. Merck, Mumbai, India).
5. Buffer 1: 0.05M sodium acetate buffer, pH 5.0.
6. Buffer 2: 0.05Msodium acetate buffer, pH 4.5.
7. Dinitrosalicylic acid reagent (10).
2.2. Entrapment of Glucoamylase and Pullulanase in Alginate Beads
1. Sodium alginate (low viscosity, Sigma).
2. Small magnetic bar.
3. Magnetic stirrer.
4. Hand-held disposable syringe, with a volume of 20 mL.
5. 22-Gauge needle.
6. 1 MCaCl2 solution.
7. Buffer 3: Buffer 1 containing 0.006 MCaCl2.
2.3. Hydrolysis of Starch in the Fluidized Bed Mode (see Note 1)
1. Jacketed glass column with a frittered end (1 x 20-cm column; Sigma).
2. Water bath with circulator.
3. Flow adapter (for 1-cm inner diameter column; Sigma).
4. Peristaltic pump with flow rates ranging from 0.5 to 20 mL/min (Alitea AB, Sweden).
5. Frac 100 fraction collector (Pharmacia, Uppsala, Sweden).
3.1. Assays of Pullulanase and Glucoamylase
1. Incubate 0.5 mL pullulanase, appropriately diluted in Buffer 1 in a reaction mixture containing 0.4% pullulan in 0.5 mL Buffer 1 at 40°C.
2. Stop the reaction after 30 min by adding 1 mL dinitrosalicylic acid reagent (10) and immersing the test tubes in a boiling water bath.
3. Cool the test tubes after boiling for 5 min, add 10 mL of distilled water, shake, and read the absorbance of the liberated reducing sugars at 540 nm. One enzyme unit liberates 1 pmol of glucose/min at 40°C, pH 5.0.
4. Incubate 0.5 mL glucoamylase, appropriately diluted in Buffer 2, in a reaction mixture containing 1% starch in 0.5 mL Buffer 2, at 65°C. Stop the reaction after 15 min by adding 1 mL dinitrosalicylic acid reagent (10) and proceed as in step 2. One enzyme unit liberates 1 pmol of glucose/min at 65°C, pH 4.5.
3.2. Entrapment of Pullulanase and Glucoamylase in Alginate Beads (see
1. Add 2.5 g alginate to 100 mL Buffer 1 and stir on a magnetic stirrer till the polymer dissolves. Add 4536 U/mL pullulanase to 15 mL of alginate solution and stir gently on a magnetic stirrer until a homogeneous solution is formed.
2. Fill a syringe with 20 mL of this solution and add it dropwise through a needle into a 100-mL solution of 100 mL CaCl2 so that beads are formed. Store the beads in this solution for 1 h and then transfer to Buffer 2 containing 0.006 M CaCl2.
3. Wash the beads repeatedly with this solution until no enzyme activity is detected in the washings.
4. The beads were kept at 4°C until further use. 458 U of glucoamylase was entrapped in a similar manner (see Note 3).
1. Pack a jacketed glass column with enzyme-entrapped alginate beads (127.5 U of glucoamylase and 85 U of pullulanase; see Note 4) and wash the beads thoroughly with Buffer 3.
2. Maintain a water bath at 45°C and circulate the water around the jacketed column. Connect tubings from the peristaltic pump to the bottom of the column.
3. Fix the adapter at the top of the column and connect the tubings out of it to the fraction collector (see Fig. 2).
4. Adjust the height of the flow adapter so that there is enough space on top of the bed for the beads to move upwards.
5. Pump the equilibrating buffer to the bottom of the column and note the increase in height of the settled bed of beads containing entrapped enzymes.
6. When the bed has fluidized to the desired height (see Note 5), pump in 1% starch from the bottom of the column at a flow rate of 2 mL/min, and collect the hydro-lyzate in a fraction collector (see Note 6).
1. Fluidized beds inherently have large void volumes. Hence high biocatalyst concentration (per unit volume of the reactor) is not possible.
2. In general, calcium alginate beads are not used for enzyme entrapment as the large pore sizes of these beads result in enzyme leakage out of these beads. However both glucoamylase and pullulanase are known to bind to alginate (11). Thus, presumably, in both cases, it is a combination of binding and entrapment that keeps the enzyme firmly held in each case.
3. The approach illustrated here is possible only if the same beads are used for the immobilization of two (or more!) enzymes (on different populations of beads!). If the beads used in the two cases are different, their fluidization behavior will be different.
4. This approach is different from two enzymes coimmobilized on the same beads. The strategy described here allows one to vary and determine optimum blend of the two immobilized enzymes in a facile manner.
5. Fluidized beds, which are "stable" and where backmixing is very low, have been termed "expanded beds" in the context of bioseparation. For a good discussion on the theory and operation of expanded beds, please consult ref. 12.
6. Comparative data about performance of fluidized bed and other reactors is rather meager. Hence it is advisable that one explores all reactor designs for process optimization. For substrate solutions containing suspended matter, fluidized bed reactors offer the clear advantage of working with such feed without any preclarification.
Financial support from Department of Science and Technology, Department of
Biotechnology and Council for Scientific and Industrial Research, all Government of India organizations, is gratefully acknowledged.
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