Enzyme immobilization, lipase

Adsorption on solid matrices, which improves (at optimal protein/support ratios) enzyme dispersion, reduces diffusion limitations and favors substrate access to individual enzyme molecules. Immobilized lipases with excellent activity and stability were obtained by entrapping the enzymes in hydrophobic sol-gel materials [20]. Finally, in order to minimize substrate diffusion limitations and maximize enzyme dispersion, various approaches have been attempted to solubilize the biocatalysts in organic solvents. The most widespread method is the one based on the covalent linking of the amphiphilic polymer polyethylene glycol (PEG) to enzyme molecules [21]. 

Different enzymes - particularly lipases - immobilized in membrane reactors have been studied in the presence of two-liquid phases (Table 5). Organic and aqueous phases containing respectively hydrophobic and hydrophilic reactants are separated by a solid mem-... 

Enzyme activity for the polymerization of lactones was improved by the immobilization on Celite [93]. Immobilized lipase PF adsorbed on a Celite showed much higher catalytic activity than that before the immobilization. The catalytic activity was further enhanced by the addition of a sugar or poly(ethylene glycol) in the immobilization. Surfactant-coated lipase efficiently polymerized the ring-opening polymerization of lactones in organic solvents [94]. 

One of the most promising applications of enzyme-immobilized mesoporous materials is as microscopic reactors. Galameau et al. investigated the effect of mesoporous silica structures and their surface natures on the activity of immobilized lipases [199]. Too hydrophilic (pure silica) or too hydrophobic (butyl-grafted silica) supports are not appropriate for the development of high activity for lipases. An adequate hydrophobic/hydrophilic balance of the support, such as a supported-micelle, provides the best route to enhance lipase activity. They also encapsulated the lipases in sponge mesoporous silicates, a new procedure based on the addition of a mixture of lecithin and amines to a sol-gel synthesis to provide pore-size control. 

In this communication a study of the catalytic behavior of the immobilized Rhizomucor miehei lipase in the transesterification reaction to biodiesel production has been reported. The main drawbacks associated to the current biodiesel production by basic homogeneous catalysis could be overcome by using immobilized lipases. Immobilization by adsorption and entrapment have been used as methods to prepare the heterogeneous biocatalyst. Zeolites and related materials have been used as inorganic lipase supports. To promote the enzyme adsorption, the surface of the supports have been functionalized by synthesis procedures or by post-treatments. While, the enzyme entrapping procedure has been carried out by sol-gel method in order to obtain the biocatalyst protected by a mesoporous matrix and to reduce its leaching after several catalytic uses. 

It was reported that PEGylated lipase entrapped in PVA cryogel could be conveniently used in organic solvent biocatalysis [279], This method for enzyme immobilization is more convenient in comparison to other types of immobilization that take advantage of enzyme covalent linkage to insoluble matrix, since the chemical step which is time consuming and harmful to enzyme activity is avoided. The application of this catalytic system to the hydrolysis of acetoxycoumarins demonstrated the feasibility of proposed method in the hydrolysis products of pharmaceutical interest and to obtain regioselective enrichment of one of the two monodeacetylated derivatives. 

The procedure shows that it is feasible to combine racemization with the kinetic resolution process (hence the DKR) of R,S)- ethoxyethyl ibuprofen ester. The chemical synthesis of the ester can be applied to any esters, as it is a common procedure. The immobilized lipase preparation procedure can also be used with any enzymes or support of choice. However, the enzyme loading will need to be optimized first. The procedures for the enzymatic kinetic resolution and DKR will need to be adjusted accordingly with different esters. Through this method, the enantiopurity of (5)-ibuprofen was found to be 99.4 % and the conversion was 85 %. It was demonstrated through our work that the synthesis of (5)-ibuprofen via DKR is highly dependent on the suitability of the reaction medium between enzymatic kinetic resolution and the racemization process. This is because the compatibility between both processes is crucial for the success of the DKR. The choice of base catalyst will vary from one reaction to another, but the basic procedures used in this work can be applied. DKRs of other profens have been reported by Lin and Tsai and Chen et al. ... 

In a like manner, a co-polymer of styrene and acryloxysuccinimide with a 10 to 1 ratio was prepared. Enzymes immobilized on this type of polymer had different physical properties. They are soluble in organic solvents such as dioxane and DMF, but insoluble in aqueous solutions. Lipases and cholesterol esterase immobilized on this type of polymer are very stable and active in several organic solvents, and have been used in several enantioselective transformations. The protocols for the immobilization are depicted in Figure 13. 

Chemoenzymatic polymerizations have the potential to further increase macro-molecular complexity by overcoming these limitations. Their combination with other polymerization techniques can give access to such structures. Depending on the mutual compatibility, multistep reactions as well as cascade reactions have been reported for the synthesis of polymer architectures and will be reviewed in the first part of this article. A unique feature of enzymes is their selectivity, such as regio-, chemo-, and in particular enantioselectivity. This offers oppormnities to synthesize novel chiral polymers and polymer architectures when combined with chemical catalysis. This will be discussed in the second part of this article. Generally, we will focus on the developments of the last 5-8 years. Unless otherwise noted, the term enzyme or lipase in this chapter refers to Candida antarctica Lipase B (CALB) or Novozym 435 (CALB immobilized on macroporous resin). 

The catalysts were found to be stable up to 323 K without any deactivation. An activation energy of 45 kJ/mol was found for the free enzyme, 41 kJ/mol for the immobilized lipase, indeed confirming the absence of mass transfer limitations. 

Lipases are extraordinary enzymes in the sense that the lipase is functioning on an interface between two phases (oil/water). This interface and the low amount of water are obtained by using immobilized lipase. The lipase is adsorbed on carriers such as anion exchange resins or adsorbers and covered with a thin film of water. 

Balcao, V.M., Paiva, A.L. and Malcata, F.X. (1996) Bioreactors with immobilized lipases State of the art. Enzyme and Microbial Technology, 18, 392-416. 

Bouwer, S.T., Cuperms, F.P. and Derksen, J.T.P. (1997) The performance of enzyme-membrane reactors with immobilized lipase. Enzyme and Microbial Technology, 21, 291-296. 

Internal diffusional limitations are possible any time that a porous immobilized enzymatic preparation is used. Bernard et al. (1992) studied internal diffusional limitations in the esterification of myristic acid with ethanol, catalyzed by immobilized lipase from Mucor miehei (Lipozyme). No internal mass diffusion would exist if there was no change in the initial velocity of the reaction while the enzyme particle size was changed. Bernard found this was not the case, however, and the initial velocity decreased with increasing particle size. This corresponds to an efficiency of reaction decrease from 0.6 to 0.36 for a particle size increase from 180 pm to 480 pm. Using the Thiele modulus, they also determined that for a reaction efficiency of 90% a particle size of 30 pm would be necessary. While Bernard et al. found that their system was limited by internal diffusion, Steytler et al. (1991) found that when they investigated the effect of different sizes of glass bead, 1 mm and 3 mm, no change in reaction rate was observed. 

Gunnlaugsdottir, H. Wannerberger, K. Sivik, B. Alcoholysis and Glyceride Synthesis with Immobilized Lipase on Controlled-Pore Glass of Varying Hydrophobicity in Supercritical Carbon Dioxide. Enzyme Microb. Technol. 1998b, 22, 360-67. 

Lipase PS-30 was immobilized on Accurel PP and the immobilized enzyme was reused five times without any loss of activity or productivity in the resolution process to prepare A-(+)-(43). The enzymatic process was scaled up to a 640-liter preparative batch using immobilized lipase PS-30 at 4 g/liter racemic substrate (43) in toluene as a solvent. From the reaction mixture, i -(+)-(43) was isolated in 35 M% overall yield with 98.5% e.e. and 99.5% chemical purity. The undesired, S -(-)-acetatc (46) produced by this process was enzymatically hydrolyzed by lipase PS-30 in a biphasic system to prepare the corresponding S -(-)-alcohol (43). Thus both enantiomers of alcohol (43) were produced by the enzymatic process. 

To understand the potential of pressure application to enzyme processes and to help elucidate the reaction mechanism as well as a rational design of alcoholysis reactors for future scale-up, we investigated the influence of temperature, pressure, exposure times, and decompression rates on the activity of a commercial immobilized lipase (Novozym 435) activity in high-pressure C02 medium. 

We conclude that a commercial immobilized lipase from C. antarctica (Novozym 435) was stable in SCC02 for all experimental conditions investigated. Based on the results obtained here and comparison of them with the results obtained by other investigators, it can be concluded that the magnitude of pressure, temperature, decompression rate, and exposure time needed to inactivate the enzyme strongly depends on the nature and the source of enzyme and, primarily, whether the enzyme is in its native or immobilized form. For the purpose of using this enzyme to catalyze the transesterification reaction of vegetable oils in order to produce esters, the results obtained herein are relevant, because the immobilized lipase can be used with low activity loss at typical conditions of temperature and pressure employed in many biotransformations of raw materials. 

Butanol conversion to butyl butyrate employing different concentrations of immobilized lipase was examined in the presence of butyric acid and n-butanol at a 1.25 1 molar ratio. Syntheses were performed at 37°C, and immobilized lipase concentration varied from 5 to 50 mg/mL. As expected, completion of the reaction was very dependent on the enzyme... 

Fig. 1. Effect of immobilized lipase contents on the butyl butyrate synthesis at 37°C for 24 h. A molar ratio between butyric acid and butanol of 1.25 was used for enzyme contents of 5,10, 25, and 50 mg/mL.

For immobilized lipase preparations, a more complex mechanism is expected to occur since esterification efficiency is also highly dependent on the hydration state of the enzyme preparation, which can be greatly modified by the nature of the substrate and the support (1,4)- In the case of butyl butyrate synthesis, analysis of substrate polarity measured as partition coefficient (Table 1) showed a higher value for butanol than for butyric acid, favoring butanol migration to the solid phase (immobilized lipase). Thus, there should be more alcohol than acid at the active site of the immobilized lipase, requiring an excess of acid in the reaction medium to provide equimolar amounts of reactants and satisfactory yields (7). 

Hydrolytic activities of free and immobilized lipase were assayed by the olive oil emulsion method according to the modification proposed by Soares et al. (11). One unit of enzyme activity was defined as the amount of enzyme that liberated 1 imol of free fatty acid/min under the assay conditions (37°C, pH 7.0,150 rpm). Analyses of hydrolytic activities carried out on the lipase loading solution and immobilized preparations were used to determine the activity-coupling yield (r %), which measures the recovered enzymatic activity according to Eq. 1 ... 

The objective of the present work was to study the synthesis of monolaurin by direct lipase-catalyzed esterification between glycerol and lauric acid without any solvent or surfactant. The effects of lauric acid/ glycerol molar ratio, enzyme concentration, and temperature were studied using an experimental design. The reuse of the commercial immobilized lipase, to reduce the process cost, was also investigated. 

An example where a transition metal catalyst is used in combination with an enzyme has been described (Scheme 19.26).207 The racemic alcohol 50 was converted to the (A1)-acetate 51, using a ruthenium catalyst along with Novozym 435 (immobilized Lipase B from Candida antarctica), 3 equivalents of p-chlorophenylacetate in t-BuOH, and 1 equivalent of 1-indanone. The reaction yield was 81% with an optical purity of >99.5% ee. 

BDF is produced currently by a chemical process with an alkaline catalyst, which has some drawbacks, such as the energy-intensive nature of the process, the interference of the reaction by free fatty acids (FFAs) and water, the need for removal of alkaline catalyst from the product, the difficulty in recovering glycerol, and the treatment of alkaline wastewater. To overcome these problems, the processes using ion-exchange resins (Shibasaki-Kitakawa et al., 2007), supercritical MeOH (Kusdiana and Saka, 2004), MeOH vapor (Ishikawa et al, 2005), and immobilized lipases (Mittelbach, 1990 Nelson et al, 1996 Selmi and Thomas, 1998) have been proposed. In this paper, enzyme processes for production of BDF from waste edible oil, waste FFAs, and acid oil recovered from soapstock are described. In addition, applications of the element reactions to the oil and fat industry are introduced. 

There were several reports that TAGs can be converted to FAMEs using an immobilized lipase. But the conversion in the reaction systems was not high, and the immobilized enzyme was not used repeatedly. Hence, we first attempted to resolve these problems. 

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