Immobilized enzyme batch

Figures 7.21 and 7.22 show typical results for the two cases-with enzymatic gel layer formation and when soluble enzymes are confined only near the membrane surface. Comprehensive models for an immobilized enzyme batch membrane reactor (IEMR) and for a soluble enzyme batch membrane reactor (SEMR) are proposed in References 33 and 30, respectively, for a flat slab membrane configuration.

A commercial pectinase, immobilised on appropriately functionalised y-alumina spheres, was loaded in a packed bed reactor and employed to depolymerise the pectin contained in a model solution and in the apple juice. The activity of the immobilized enzyme was tested in several batch reactions and compared with the one of the free enzyme. A successful apple juice depectinisation was obtained using the pectinase immobilised system. In addition, an endopolygalacturonase from Kluyveromyces marxianus, previously purified in a single-step process with coreshell microspheres specifically prepared, was immobilised on the same active support and the efficiency of the resulting catalyst was tested. 

Purified MeHNL was crystallized by the sitting-drop vapor-diffusion method. The 10-20 mm bipyramidal crystals formed were cross-linked with glutaraldehyde and used as biocatalyst for the synthesis of optically active cyanohydrins. The cross-linked crystals were more stable than Celite-immobilized enzymes when incubated in organic solvents, especially in polar solvents. After six consecutive batch reactions in dibutyl ether, the remaining activity of the cross-linked crystals was more than 70 times higher than for the immobilized enzymes. Nevertheless, the specific activity of the cross-linked crystals per milligram protein was reduced compared with the activity of Celite-immobilized enzymes [53],... 

In summary, the synthesis and in situ regeneration of nucleotide sugars by combinatorial biocatalysis suffers from the main disadvantage that each enzyme has to be produced in sufficient amounts. This affords efficient recombinant protein produchon hosts being a bottleneck for some genes [25]. However, once a multi-enzyme system has been developed, the productivity can be improved by repetitive use of the biocatalysts as demonstrated for repetitive batch syntheses with soluble enzymes [25, 38] or with immobilized enzymes [48]. The advantage... 

Milk treatment tends to be confined to batch processes since continuous processing with immobilized enzyme results in microbial contamination, which is encouraged because mild conditions of temperature and pH have to be used. 

The first cross-linked HNLs were reported by Costes et al. [72]. They compared MeHNL-CLECs with Celite -immobilized MeHNL. By cross-linking, the stability of the enzyme was improved, particularly in polar organic solvents. Furthermore, the cross-linked crystals could be reused without significant loss of activity. After six consecutive batches, 70% of the initial activity was retained, whereas the immobilized enzyme showed virtually no remaining activity (<1%). Nevertheless, crystallization and cross-linking cause a considerable loss of activity compared to the immobilization on Celite [72]. 

Enzymes, when immobilized in spherical particles or in films made from various polymers and porous materials, are referred to as immobUized enzymes. Enzymes can be immobilized by covalent bonding, electrostatic interaction, crosslinking of the enzymes, and entrapment in a polymer network, among other techniques. In the case of batch reactors, the particles or films of immobilized enzymes can be reused after having been separated from the solution after reaction by physical means, such as sedimentation, centrifugation, and filtration. Immobilized enzymes can also be used in continuous fixed-bed reactors, fluidized reactors, and membrane reactors. 

Immobilized enzyme beads with a diameter of 10 mm containing the same amount of the enzyme above are used in the same stirred-batch reactor. Determine the initial reaction rate of the substrate solution of 0.1 kinolm". Assume that the effective diffusion coefficient of the substrate in the catalyst beads is 1.0 x 10" cm s". ... 

The number of relevant applications, especially those dealing with immobilized enzyme reactors, increases steadily (267, 268). Immobilization of enzymes on suitable matrices permits their reuse, thus creating the possibility to perform more experiments with the same batch of enzymes, which cuts down the cost of the analysis. 

Mensah, P. Gainer, J. L. Carta, G. Adsorptive Control of Water in Esterification with Immobilized Enzymes I. Batch Reactor Behavior. Biotechnol. Bioeng. 1998, 60 (4), 434-444. 

There is some differences in literature reports regarding the optimum pH for the enzymatic action of SOD. MacLeod et al (2Q) and Cohen and Fridovich (21) report optimum pH levels of 8.5-8.6 while Smith (2) found best results at pH 7 for enzyme-coated electrodes. For the immobilized eimane, Masoom and Townshend ( ) reported optimum performance between pH 8.5 and 9.0. Our results, shown in Figure 5b, largely confirms the original reports (30.31) in these experiments, the buffer composition in the peroxidase reagent were altered to maintain the same pH for the second enzymatic reaction step. We have subsequently found however, that the quality and the behavior of the enzyme purchased in different batches can vary significantly. At least one enzyme batch performed optimally at a pH substantially lower than pH 8.5, lending credence to the report of Smith (2). 

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. 

Batch studies for evaluating immobilized enzyme activity and properties of the "bioplastic" (urease entrapped in PDMS) material were conducted in 250-mL shake flasks in an environmentally controlled shaker/ incubator. 

The hydrolysis of an IV-acylated amino acid by an enzyme provides a resolution method to amino acids. Because the starting materials are readily available in the racemic series by the Schotten-Baumann reaction, the method can be cost effective (Scheme 2.21).68-71 The L-amino acid product can be separated by crystallization, whereas the D-amino acid, which is still /V-acylated, can be recycled by being resubjected to the Schotten-Baumann conditions used for the next batch. Tanabe has developed a process with an immobilized enzyme,72 73 whereas Degussa uses the method in a membrane reactor.69 74 The process is used to make L-methionine. 

It is well known that immobilization techniques may improve enzyme stability and have a protective effect against inactivation [7]. Moreover, immobilized enzymes are easy to recover and reuse at the end of a discontinuous process. Immobilized peroxidases protected against peroxide inactivation were extensively applied in batch systems (see Chap. 9) [7, 20, 37, 59, 78, 95]. 

To measure the activity in transesterification, equimolar amounts of triolein and tripalmitin were reacted by means of immobilized enzyme (8). The transesterification was followed by HPLC analysis of the triglycerides. One Batch Transesterification Unit is the initial exchange of fatty acids in jtcmol per min. 

Immobilized enzymes can be used in one of two basic types of reactor systems. The first is the stirred tank reactor where the immobilized enzyme is stirred with the substrate solution. This is a batch system and, after the reaction is complete, the immobilized enzyme is separated from the product. The other system employs continuous flow columns in which the substrate flows through the immobilized enzyme contained in a column or similar device. A simplified flow diagram of such a system is given in Figure 10-23. 

Free enzyme versus immobilized enzyme can influence the yield of lOS, additionally an immobilized system would be favorable economically as the biocatalyst can be reused, enables continuous production and the end product is free of contamination. Kim et al. [275] intended to make a comparison between the reaction kinetics of free and immobilized endo-inulinases in a batch reactor however significant differences were observed in the reaction behavior and product composition due to the form of enzyme used and the initial concentration of substrate. Yun et al. [276] investigated the effect of inulin concentration on the production of lOS by free and immobilized endo-inulinase from Pseudomonas sp. Their findings corroborate those of Kim et al. [275] whereby different products are formed depending on the form of enzyme a soluble enzyme yielded inulobiose and DP3 products, whereby the immobilized form predominantly produced inulobiose. As the concentration of inulin increased the yield of lOS did not increase in the soluble system and in the immobilized the yield remained the same. Although the enzyme was derived from Pseudomonas the immobilized form required a differ-... 

The isomerization of the glucose syrup thus obtained was originally also employed in batch reactors using the Mg -containing enzyme glucose isomerase in soluble form, but rapidly evolved to the use of an immobilized enzyme... 

Because the costs of isolation and purification of soluble enzymes are high and it is often both technically difficult and costly to recover an active form of the enzyme from product mixtures when the reaction of interest is completed, soluble enzymes are normally employed only in batch operations in which the enzymes are removed from the liquid product by precipitation. Thermal deactivation may be used instead to destroy the catalytic activity of the enzyme. Immobilization of the enzyme circumvents these difficulties because the solid phase containing the enzyme is easily recovered from the product mixture. Use of immobilized enzymes makes it possible to conduct the process in a continuous flow mode, thereby facilitating process control via manipulation of the flow rate of the process stream. One can offset losses in enzyme activity as time elapses by reducing the flow rate to maintain a constant product composition. Operation in this mode permits one to obtain more product per unit of enzyme employed. 

The catalytic behavior of enzymes in immobilized form may dramatically differ from that of soluble homogeneous enzymes. In particular, mass transport effects (the transport of a substrate to the catalyst and diffusion of reaction products away from the catalyst matrix) may result in the reduction of the overall activity. Mass transport effects are usually divided into two categories - external and internal. External effects stem from the fact that substrates must be transported from the bulk solution to the surface of an immobilized enzyme. Internal diffusional limitations occur when a substrate penetrates inside the immobilized enzyme particle, such as porous carriers, polymeric microspheres, membranes, etc. The classical treatment of mass transfer in heterogeneous catalysis has been successfully applied to immobilized enzymes I27l There are several simple experimental criteria or tests that allow one to determine whether a reaction is limited by external diffusion. For example, if a reaction is completely limited by external diffusion, the rate of the process should not depend on pH or enzyme concentration. At the same time the rate of reaction will depend on the stirring in the batch reactor or on the flow rate of a substrate in the column reactor. 

A plug flow reactor may be realized using immobilized enzymes within a column reactor or using soluble enzymes within a cascade of membrane reactors. A batch or a repetitive batch process with soluble enzymes (see below) has the same productivity as the plug flow reactor. 

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