Immobilization supports Novozym

Figure 32 includes results illustrating the performance of lipase/car-bon monolith systems in an acylation reaction. For comparison, the free lipase and a commercial immobilized lipase (Novozyme) were also tested. As expected, in all cases the specific activity of immobilized lipase was foimd to be lower than that of the free enzyme. Such a difference is usually ascribed to conformational changes of the enzyme, steric effects, or denaturation. For the monolithic biocatalysts, the activity of the immobilized catalyst relative to that of the pure enzyme was found to be 30-35%, and for the Novozyme catalyst about 80% in the first rim. However, the Novozyme catalyst underwent significant deactivation, in contrast to the carbon monolith-supported catalysts. The deactivation of the Novozyme catalyst in consecutive runs is probably a consequence of the instability of the support matrix under reaction conditions (101,102). 

FIGURE 32 Initial rates of reaction in catalysis by free lipase and immobilized lipase (Novozyme, and catalyst supported on 200 cpsi carbon monolith) in the acylation of butanol with vinyl acetate in an organic medium at 300 K. 

Experiments were typically performed at 70°C in toluene (125 mL) in a glass reactor at atmospheric pressure under H2 flow (AGA 99.999, 295 mL/min). The initial reactant concentration was 0.02 mol/L. Ethyl acetate with the concentration of 0.06 mol/L was used as an acyl donor. The catalytic hydrogenation of acetophenone (Acros, 99%) was carried out over 2% (w/w) Pd/N-CNT (312.5 mg) and the formed / -l-phenylethanol was acylated in the same pot to / -l-phenylethyl acetate with an immobilized lipase (Novozym 435, lipase B from Candida antarctica) (62.5 mg). The supported Pd catalysts were pre-reduced at 200 °C prior to the experiment. 

In conventional synthetic transformations, enzymes are normally used in aqueous or organic solvent at moderate temperatures to preserve the activity of enzymes. Consequently, some of these reactions require longer reaction times. In view of the newer developments wherein enzymes can be immobilized on solid supports [183], they are amenable to relatively higher temperature reaction with adequate pH control. The application of MW irradiation has been explored with two enzyme systems namely Pseudomonas lipase dispersed in Hyflo Super Cell and commercially available SP 435 Novozym (Candida antarctica lipase grafted on an acrylic resin). 

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). 

Enzymatic resolution of racemic functionalized isoxazolines is a valuable technique for the preparation of enantio-merically enriched and pure 4,5-dihydroisoxazoles. These enzymatic resolutions exploit a variety of transformations of functional groups resident on the side-chains of the isoxazoline ring. The multipolymer enzymatic resolution of soluble polymer-supported alcohols 307 and 308 was achieved using an immobilized lipase from Candida antarctica (Novozym 435). The (R)-alcohol 309 was obtained in enantiomerically pure form (>99% ee) after its cleavage from the poly(ethylene glycol) (PEG) scaffold (Scheme 70) <2000JOG8527>. 

Lipases act in nature on oil-water interfaces and often have hydrophobic domains on their surface. Hence, immobilization by adsorption to a hydrophobic carrier is often a simple and effective way to immobilize Upases. A wide range of different hydrophobic support materials is commercially available, including synthetic acrylic, divinylbenzene-styrene or polypropylene polymers. An example of the latter is Accurel MP 1000, which is available from Membrana. Novozym 435 is immobilized on Lewatit VP OC 1600, a divinylbenzene-cross-linked poly(methyl methacrylate) resin produced by Lanxess (previously Bayer). 

The molecular weight of PCL polymerized using four lab-scale immobilized enzymes are shown in Figure 3.9. The PLU values were also measured. The results are compared with Novozym 435 and NS81018. Both PLU values and the molecular weights show that none of the lab-scale immobilized samples could reach the activity of Novozym 435. Even when Lewatit was used as a support,... 

Abstract An agroindustrial residue, green coconut fiber, was evaluated as support for immobilization of Candida antarctica type B (CALB) lipase by physical adsorption. The influence of several parameters, such as contact time, amount of enzyme offered to immobilization, and pH of lipase solution was analyzed to select a suitable immobilization protocol. Kinetic constants of soluble and immobilized lipases were assayed. Thermal and operational stability of the immobilized enzyme, obtained after 2 h of contact between coconut fiber and enzyme solution, containing 40 U/ml in 25 mM sodium phosphate buffer pH 7, were determined. CALB immobilization by adsorption on coconut fiber promoted an increase in thermal stability at 50 and 60 °C, as half-lives (t /2) of the immobilized enzyme were, respectively, 2- and 92-fold higher than the ones for soluble enzyme. Furthermore, operational stabilities of methyl butyrate hydrolysis and butyl butyrate synthesis were evaluated. After the third cycle of methyl butyrate hydrolysis, it retained less than 50% of the initial activity, while Novozyme 435 retained more than 70% after the tenth cycle. However, in the synthesis of butyl butyrate, CALB immobilized on coconut fiber showed a good operational stability when compared to Novozyme 435, retaining 80% of its initial activity after the sixth cycle of reaction. 

The thermal stability at 60 °C of Novozyme 435, however, is higher than that of CALB-7A (Fig. 5). After 10 h of incubation at 60 °C, Novozyme 435 retained more than 70% of its initial activity, whereas CALB-7A retained only 50%. The higher stability of Novozyme 435 may be due to the hydrophobic nature of the support used for immobilization. When hydrophobic supports are used, the hydrophobic areas surrounding the enzyme active center are involved in adsorption, which stabilizes the active form of lipase [11]. 

Reusability of immobilized CALB was tested in subsequent cycles of methyl butyrate hydrolysis. It can be observed in Fig. 7 that CALB-7A retained less than 50% of its initial hydrolytic activity after the third cycle of reaction whereas Novozyme 435 retained almost 70% after the tenth cycle (Fig. 7). Other authors [36] observed that CALB immobilized on activated carbon retained more than 55% of its initial activity after the sixth cycle of methyl butyrate hydrolysis. The worse operational stability of CALB immobilized on coconut fiber, when compared to CALB immobilized on activated carbon and to Novozyme 435, may be due to enzyme desorption during reaction, induced by the hydrophobic substrate, and by the low enzyme load adsorbed. As discussed before, the driven forces of CALB adsorption on coconut fiber are electrostatic interactions that are weaker than hydrophobic interactions, which predominate on Novozyme 435 and CALB adsorbed on activated carbon. Furthermore, both aetivated carbon and the resin used in the preparation of Novozyme 435 are porous support with high superficial area available for enzyme immobilization, allowing obtaining of high enzyme load. Coconut fiber, on the other hand, does not have a porous structure, and it has a low surface area [27], making it difficult to achieve high enzyme loads. 

In the present work, green coconut fiber was successfiilly used to immobilize lipase B from C. antarctica by adsorption. During adsorption studies, it was observed that adsorption equilibrium was achieved afler a contact time of 2 h (in case of o=30 U/ml and Eo=(0 U/ml) or 6 h ( o=90 U/ml). Moreover, an improvement of hydrolytic activity of immobilized CALB is also observed with increasing concentrations of lipase offered to immobilization. This increase in activity is due to formation of multilayers, confirmed by thermal stability essays. Two plateaus of enzyme activity were observed when the pH of lipase solution during adsorption was varied in the range studied. This behavior is typical of an ionic support At 50 and 60 °C, the adsorbed enzyme was, respectively, 2- and 92-fold more stable than the soluble enzyme. At 60 °C, however, Novozyme 435 s stability was higher than that of CALB-7A. TUIer 10 h of incubation at 60 °C, Novozyme 435 retained more than 70% of its initial activity, whereas CALB-7A retained only 50%. Last but not least operational stabilities studies of butyl butyrate synthesis, compared to a commercial derivative, showed that C7U..B-7A is a suitable biocatalyst to be used in the synthesis of flavors. 

Candida antarctica Lipase B (CALB) is atfracting increasing attention as a biocatalyst for the synthesis of low molar mass and polymeric molecules. Almost all publications on immobilized CALB use the commercially available catalyst Novozym 435, which consists of CALB physically adsorbed onto a macroporous acrylic polymer resin (Lewatit VP OC 1600, Bayer). Primarily, commercial uses of CALB are limited to production of high-priced specialty chemicals because of the high cost of commercially available CALB preparations Novozym 435 (Novozymes A/S) and Chirazyme (Roche Molecular Biochemicals). Studies to better correlate enzyme activity to support parameters will lead to improved catalysts that have acceptable price-performance characteristics for an expanded range of industrial processes. 

This work was partially supported by a Grant-in-Aid for General Scientific Research and by a Grant-in-Aid for the 21st Century COE Program KEIO LCC from the Ministry of Education, Culture, Sports, Science, and Technology, Japan. Immobilized lipase from Candida antarctica (CA, Novozym 435) was kindly supplied by Novozymes Japan Ltd. (Chiba, Japan). 

The most commonly used hydrolase for biocatalysis is lipase B from C. antarctica (CAL-B). The commercial material is usually Novozym 435, which is protein immobilized noncovalently on an acrylic resin. This immobilization is suitable for use in organic solvents, but in water, the lipase desorbs from the support. CAL-B is a recombinant protein from the yeast C. antarctica produced in a strain of the fungus Aspergillus oryzae [5]. CAL-B shows little or no interfacial activation and hydrolyzes long chain triglycerides only slowly. For this reason, it may be better classified as an esterase It shows high activity and enantioselertivity toward a... 

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