Biocatalysts enzyme immobilization

Most industrial enzymatic processes refer to reactions conducted by hydrolases in aqueous medium for the degradation of complex molecules (often polymers) into simpler molecules in conventional processes with limited added value (Neidelman 1991). Reasons underlying are clear since hydrolases are robust, usually extracellular and have no coenzyme requirements, which makes them ideal process biocatalysts. Enzyme immobilization widened the scope of application allowing less stable, intracellular and non-hydrolytic enzymes to be developed as process biocatalysts (Poulsen 1984 D Souza 1999), as illustrated by the paradigmatic case of glucose isomerase for the production of HFS (Carasik and Carroll 1983) and the production of acrylamide from acrylonitrile by nitrile hydratase (Yamada and Kobayashi 1996). 

Examples of immobilized biocatalysts. Enzyme immobilization clearly imparts many benefits to biocatalysts. A primary advantage is the opportunity to tailor an immobilization matrix for a specific application, use, or set of conditions. Such a process has been, and continues to be, undertaken for a host of biocatalysts and a broad array of applications. The authors describe herein one instance of adapting immobilization techniques and chemistry to a specific application of biocatalysis. Although the specifics and details of this particular effort may not be applicable to every and all uses of enzyme catalysis, some detail is provided to convey to the reader those issues that need to be considered when one attempts to immobilize enzymes for a particular task. 

The biotransformation process has been improved by significant advances in biochemical engineering advances in genetic and protein engineering, microbiological manipulations for the production of enzymes, and the use of biocatalysts in immobilized form and large-scale purification methods. 

Ballesteros et defined immobilized biocatalysts as enzymes, cells or organelles (or combinations of these) which are in a state that permits their reuse . Enzyme immobilization represents only a small part of this field, but is the most commonly employed in pharmaceutical production. 

Ballesteros, A., van Beynum, G., Bomd, O. and Buchholz, K., Guidelines for the characterization of immobilized biocatalysts. Enzyme Microb. TechnoL, 1983, 5, 304-307. 

For some recent reviews, see Pfenosil, J.E., Kut, O.M., Dunn, I.J. and Heinzle, E., Immobilized biocatalysts. In Ullman s Biotechnology and Biochemical Engineering, vol. 2. Wiley-VCH, Weinheim, 2007, pp. 683-734 Sheldon, R. A., Enzyme immobilization the quest for optimum performance. Adv. Synth. Catal., 2007, 349, 1289-1307 End, N. and Schoning, K.-U., Immobilized biocatalysts in industrial research and production. Topics Curr. Chem., 2004, 242, 273-317 Bornscheuer, U.T., Immobilizing enz3mies how to create more suitable biocatalysts. Angew. Chem. Int. Ed., 2003,42, 3336-3337 Cao, L. Immobilised enzymes science or art Curr. Opin. Chem. Biol., 2005, 9, 217-226. 

The recent literature in bioelectrochemical technology, covering primarily the electrochemical aspects of enzyme immobilization and mediation, includes few reports describing engineering aspects of enzymatic biofuel cells or related devices. Current engineering efforts address issues of catalytic rate and stability by seeking improved kinetic and thermodynamic properties in modified enzymes or synthesized enzyme mimics. Equally important is the development of materials and electrode structures that fully maximize the reaction rates of known biocatalysts within a stable environment. Ultimately, the performance of biocatalysts can be assessed only by their implementation in practical devices. 

The use of enzymes as biocatalysts for the synthesis of water-soluble conducting polymers is simple, environmentally benign, and gives yields of over 90% due to the high efficiency of the enzyme catalyst. Since the use of an enzyme solution does not allow the recovery and reuse of the expensive enzyme, well-established strategies of enzyme immobilization onto solid supports have been applied to HRP [22-30]. A recent work reported an alternative method that allows the recycle and reuse of HRP in the biocatalytic synthesis of ICPs. The method is based on the use of a biphasic catalytic system in which the enzyme is encapsulated by simple solubilization into an IL. The main strategy consisted of encapsulating the HRP in room-temperature IPs insoluble in water, and the other components of the reaction... 

The performance of immobilized biocatalyst (enzyme) reactors is influenced by enzyme inactivation during operation, mainly due to thermal denaturation, desorption of the biocatalyst from the solid support, disintegration or solubilisation of the support and microbial attack. 

I. Chibata, T. Tosa u. T. Sato, Immobilized Biocatalysts to Produce Amino Acids and Other Organic Compounds, Biotcchnol. Ser. 1985, 5,h Enzymes Immobilized Cells Biotechnol. 37-70. 

Immobilization onto a solid support, either by surface attachment or lattice entrapment, is the more widely used approach to overcome enzyme inactivation, particularly interfacial inactivation. The support provides a protective microenvironment which often increases biocatalyst stability, although a decrease in biocata-lytic activity may occur, particularly when immobilization is by covalent bonding. Nevertheless, this approach presents drawbacks, since the complexity (and cost) of the system is increased, and mass transfer resistances and partition effects are enhanced [24]. For those applications where enzyme immobilization is not an option, wrapping up the enzyme with a protective cover has proved promising [21]. 

R Bahulekar, NR Ayangar, S Ponrathnam. Polyethyleneimine in immobilization of biocatalysts. Enzyme Microb Technol 13 858-868, 1991. 

Figure 14.2 Membrane bioreactor with immobilized biocatalysts (enzyme or micro-organism).

Asymmetric hollow fibers provide an interesting support for enzyme immobilization, in this case the membrane structure allows the retention of the enzyme into the sponge layer of the fibers by crossflow filtration. The amount of biocatalyst loaded, its distribution and activity through the support and its lifetime are very important parameters to properly orientate the development of such systems. The specific effect that the support has upon the enzyme, however, greatly depend upon both the support and the enzyme involved in the immobilization as well as the method of immobilization used. 

To test the reusability of the biocatalyst, five sequential reaction cycles with CPO immobilized on SBA-16 of different pore sizes were completed [6]. The authors found that immobilization on material with larger pore, 143 A, improved the reusability of the catalyst. Enzyme immobilized by covalent attachment to silica-based materials retained a higher residual activity after five reaction cycles than the physical approach. 

Aoun S, Chebli C, Baboulene M (1998) Noncovalent immobilization of chloroperoxidase onto talc catalytic properties of a new biocatalyst. Enzyme Microb Technol 23 380-385... 

For practical photoinduced synthetic biocatalyzed transformations, it is important to integrate biocatalysts in immobilized matrices that allow the recycling of the photosystems. The fact that bipyridinium sites act as electron mediators for various redox enzymes was used to develop two paradigms for the electrical contacting and photoactivation of the biocatalyst (Figure 39). By one approach, the bipyridinium electron relays are tethered by covalent bonds to the protein backbone (Figure 39A). These electron relays act as oxidative quenchers of the excited state of the dye and, upon photoreduction of the electron acceptor units, they act as electron carriers that activate the reductive functions of the enzyme. As an example, the... 

Flow microcalorimetry, which makes many rapid and accurate measurements of the activity of immobilized biocatalysts, provides a tool for researchers that can be used to discriminate between different preparatives of immobilized biocatalysts. Table 3 shows previous experiments where the characterization of kinetic properties by flow micro calorimetry was used to compare different techniques of purified enzyme immobilization [27, 30, 31, 35] as well as the immobilization of enzymes fixed in cells [28,29,40]. More details can be found in our recent review article [41]. 

Enzymes are characterized by unusual specific activities and remarkably high selectivities. They are effective catalysts at relatively low temperatures and ambient pressure. The primary driving force for efforts to develop immobilized forms of these biocatalysts is cost, especially when one is comparing process alternatives involving either conventional inorganic catalysts or soluble enzymes. Immobilization can permit conversion of labile enzymes into forms appropriate for use as catalysts in industrial processes—production of sweeteners, pharmaceutical intermediates, and fine chemicals—or as biosensors in analytical applications. Because of their high specificities, immobilized versions of enzymes are potentially useful in situations where it is necessary to obtain high yields of the desired product... 

Multiple authors (1983) The Working Party on Immobilized Biocatalysts Enzyme Microb Technol 5 304... 

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