A-Amylase immobilization

A membrane cell recycle reactor with continuous ethanol extraction by dibutyl phthalate increased the productivity fourfold with increased conversion of glucose from 45 to 91%.249 The ethanol was then removed from the dibutyl phthalate with water. It would be better to do this second step with a membrane. In another process, microencapsulated yeast converted glucose to ethanol, which was removed by an oleic acid phase containing a lipase that formed ethyl oleate.250 This could be used as biodiesel fuel. Continuous ultrafiltration has been used to separate the propionic acid produced from glycerol by a Propionibacterium.251 Whey proteins have been hydrolyzed enzymatically and continuously in an ultrafiltration reactor, with improved yields, productivity, and elimination of peptide coproducts.252 Continuous hydrolysis of a starch slurry has been carried out with a-amylase immobilized in a hollow fiber reactor.253 Oils have been hydrolyzed by a lipase immobilized on an aromatic polyamide ultrafiltration membrane with continuous separation of one product through the membrane to shift the equilibrium toward the desired products.254 Such a process could supplant the current energy-intensive industrial one that takes 3-24 h at 150-260X. Lipases have also been used to prepare esters. A lipase-surfactant complex in hexane was used to prepare a wax ester found in whale oil, by the esterification of 1 hexadecanol with palmitic acid in a membrane reactor.255 After 1 h, the yield was 96%. The current industrial process runs at 250°C for up to 20 h. 

Shewale, S. D., and Pandit, A. B. (2007). Hydrolysis of soluble starch using bacillus lichenifonnis a amylases immobilized on super porous celbeads, 342(8), 997-1008. 

Cho E.J., TaoZ., Tehan E.C., Bright F.V. Multianalyte pin-printed biosensor arrays based on protein-doped xerogels. Anal. Chem. 2002 74 6177-6184 Cho G., Moon I.S., Lee J.S. Preparation and characterization of a-amylase immobilized inor-ganic/organic hybrid membrane using chitisan as a dispersant in the sol-gel process. Chem. Letters 1997 577-578... 

Bacillus subtilis a-amylase immobilized on a phenol-formaldehyde resin displayed exo-activity, which might result from steric hindrance between the immobilized enzyme and substrates of high molecular weight (e.g. amylopectin). Thus, cleavage of the peripheral D-glucosidic linkages produced small oligosaccharides only. 

Fig. 24. Kinetics of add inactivation of a-amylase (Bac. subtilis) in solution (/, 2) and immobilized on Biocarb (3) 1) pH 2 2) and 3) pH 4. A/A0 is the value of relative enzymatic activity (compared to the initial activity A0 before inactivation), substrate — starch...

Fig. 25. Reactivation of inactivated of 1) a-amylase at different pH values of the solution, 2) oc-amylase immobilized in Biocarb...

Immobilization of a-Amylase on Magnetic Nanoparticles for Enhanced Reusability in Bio-Catalytic Processes... 

The decomposition of polymer molecules in wastewater samples can also be achieved by a hybrid sensor, which consists of a consortium of microorganisms and hydrolases together. As an example, the BOD of lactose-containing wastewater was determined by a hybrid sensor consisting of cells of the yeast Lipomyces kononenkoae and of the enzyme /1-galactosidase [50, 69]. It is also possible to use a column containing immobilized enzymes, which is inserted in the measuring device (e.g., an FIA) before the biosensor [52, 70]. Such a combination of an enzyme column, which contains a-amylase and amy-... 

The performance of cellulase and amylase immobilized on siliceous supports was investigated. Enzyme uptake onto the support depended on the enzyme source and immobilization conditions. For amylase, the uptake ranged between 20 and 60%, and for cellulase, 7-10%. Immobilized amylase performance was assessed by batch kinetics in 100-300 g/L of com flour at 65°C. Depending on the substrate and enzyme loading, between 40 and 60% starch conversion was obtained. Immobilized amylase was more stable than soluble amylase. Enzyme samples were preincubated in a water bath at various temperatures, then tested for activity. At 105°C, soluble amylase lost -55% of its activity, compared with -30% loss for immobilized amylase. The performance of immobilized cellulase was evaluated from batch kinetics in 10 g/L of substrate (shredded wastepaper) at 55°C. Significant hydrolysis of the wastepaper was also observed, indicating that immobilization does not preclude access to and hydrolysis of insoluble cellulose. 

Enzymes were immobilized onto silica gel by covalent crosslinking with glutaraldehyde in a procedure similar to that of Kondo et al. (3). Briefly, 1-4 g of silica gel was incubated in 1-13 wt% glutaraldehyde and in 100-1000 mL of enzyme solution for up to 48 h. The immobilized enzyme was recovered by filtration and washed to remove loosely bound enzyme. Samples of the soluble enzyme were collected before and after immobilization and assayed for activity, to provide an estimate of enzyme uptake onto the support. The a-amylases studied were Spezyme Fred (Genencor), Allyzme (Alltech), and Liquozyme (Novozymes). The cellulases studied were Spezyme CP and Spezyme CE (Genencor). 

The enzyme uptake varied significantly according to the enzyme source, type, loading, and incubation time. The typical uptake for amylase ranged between 20 and 60%, whereas for cellulase, only 7-10% uptake was obtained. Since these uptake measurements simply reflect losses in soluble enzyme activity from the immobilization solution, they set a maximum on the activity that may be expressed by the immobilized enzyme. The actual activity is expected to be less, once losses resulting from shear, conformational changes, and nonproductive binding are taken into account. Direct measurements of immobilized enzyme activity therefore provide the best measure of the effectiveness of a particular immobilization technique. 

In treating some aqueous products, such as milk or eggs, some residual cyclodextrin might remain in the product. The residual cyclodextrin can be removed, if desired, by treating the product with a cyclodextrin transglycosylase or an a-amylase.82 If an immobilized enzyme is used, the enzyme can be removed from the product. When oils are treated with cyclodextrin, undetectable levels, if any, of cyclodextrin remain in the oil phase if the proper proportions of oil and cyclodextrin solution are used. 

Fig. 7. Activity retention as a function of monomer concentration in the immobilized enzymes by radiation polymerization method. Enzyme a-amylase(— 24 °C, 1 x 106 rad) glucose isomer-ase (—45°C, 1 x 106 rad) O a-glucosidase (—78°C, 1 x 106 rad) A glucoamylase (—78CC, 1 x 106 rad). Monomer HEMA (2-hydroxyethyl methacrylate)...

Another fruitful area of research has been that of the sonochemical activation of immobilized enzymes where ultrasound appears to be particularly useful in increasing the transport of substrate to the enzyme. Using a-chymotrypsin (on agarose gel) and casein as substrate, a two-fold increase in activity was observed at 20 kHz [12]. Here the origin of the enhancement was thought to be associated with increased penetration of the casein into the support gel induced by cavitational effects close to the surface. However an increase in the activity of a-amylase (on porous polystyrene) was produced on irradiation with 7 MHz ultrasound [13]. This is a very significant result since at this high-frequency cavitation cannot occur and... 

---