Biocatalysts immobilization

A fixed bed or slurry bioreactor incorporates the biocatalyst immobilized on a solid support in an aqueous solution, mineral nutrients and an assimilable source of carbon. 

Figure 3.24 depicts a piezoelectric sensor consisting of two oscillator circuits a detector crystal oscillator and a reference crystal oscillator. The two are identical except for the fact that the reference oscillator is not coated with biological material and is intended to correct for temperature and humidity fluctuations, as well as other interfering effects. The two oscillator frequencies are fed to a mixer that provides the difference in frequency between the two crystals. In order to use the piezoelectric effect to detect a target dissolved substrate it should be reacted with a suitable biocatalyst immobilized on the crystal by entrapment (deposition from an acrylamide solution), cross-linking, irradiation or pre-coating. 

Thermal and pH sensitive heterogeneous copolymer hydrogels which contain silicone rubber domains within a temperature and pH sensitive copolymer of NIPA and acrylic acid have been synthesized by Dong et al. [60]. These materials contained macropores when swollen and collapsed much faster than homopolymers of iV-isopropylacrylamide. Biocatalyst immobilization using copolymers of NIPA and NN - dimethylaminopropylmethacrylamide have also been studied [61]. 

Ban, K., Hama, S., Nishizuka, K., Kaieda, M., Matsumoto, T., Kondo, A., Noda, H., and Fukuda, H. 2002. Repeated use of whole-cell biocatalysts immobilized within biomass support particles for biodiesel fuel production./. Mol. Catal. B Enzym., 17, 157-165. 

There has been considerable effort directed toward the immobilization of both enzymes and whole cells in a wide array of formats.15 Initial attempts to immobilize enzymes on naturally derived supports such as charcoal were conducted early in the twentieth century and eventually led to the development of more robust biocatalysts immobilized on synthetic resins by the mid-1950s. Immobilization often confers a number of advantages relative to the free biocatalyst including ease of removal from the process stream, potential for reuse, improvements in stability, favorable alterations in kinetic parameters, suitability for continuous production and in some cases the ability to operate in organic solvents. The focus of this section is on the immobilization of enzymes, however, many of the same principles apply to whole cells, the primary difference being the fact that immobilized cells are often less stable than individual enzymes and may contain additional undesired enzyme activities. 

Immobilized enzymes are not restricted to bioanalytical applications. Increasingly they attract a huge amount of interest in industrial organic chemistry due to their excellent stereo - and enantioselectivity. Moreover, they work under mild conditions of temperature, pH and pressure. Therefore, the determination of kinetic constants is of great interest. They allow quantitative characterization of immobilized biocatalyst preparations and facilitate comparisons between different materials and procedures for biocatalyst immobilization. 

Turner MB, Spear SK, Holbery JD et al (2005) Ionic liquid-reconstituted cellulose composites as solid support matrices for biocatalyst immobilization. Biomacromolecules 6 2497-2502... 

The biocatalytic process takes place in stirred tank reactors with sieves in the bottom and a working volume of several cubic meters (Fig. 7). The first biocatalytic step is a three-phase reaction with a solid biocatalyst, immobilized DAO on a spherical carrier, the liquid Ceph C solution and oxygen or air as the gaseous phase. The second step is a two-phase reaction with immobilized GA on a solid carrier and the glutaryl-7-ACA in solution. 

Opportunity for innovation and creativity still exists in the field of biocatalyst immobilization. Despite the tremendous volume of biocatalyst immobilization literature, there is no one technology that is universally applicable and no one technique that can be applied using a generic procedure. The limitations of individual immobilization techniques have been pointed out in each section. Operationally simple adsorption methods often are limited by the lack of stabilization and by protein leaching, especially under aqueous conditions. Restriction of diffusion can be severe for entrapped proteins and cells. Covalent methods often result in protein inactivation and a much higher carrier cost. The combined effects of... 

Those systems can contribute in general to improvements in methods for recovery and reuse of enzymes or whole cells development in methods for biocatalyst immobilization development of enzyme membrane reactors. 

Fruit juice clarification by pectinases and cellulases is another interesting application. In the conventional process after the enzymatic reaction in the pulp treatment step takes place, filtration over diatomaceous earth follows. This filtration-type process produces a lot of solid waste, and results in costly enzyme loss. MBR are appropriate for such application either for enzyme recovery and recycle or in the form of a more compact CMR type system, with the biocatalyst immobilized on the membrane itself [4.58]. 

Adsorption is a simple and straightforward route for biocatalyst immobilization which offers unique advantages over soluble enzymes, such as enhanced activity, increased selectivity, improved stability and reusability. For example, Assemblase, the commercial name of immobilized pencillin-G acylase from E. coli, has been used by industry for manufacture of the semi-synthetic p-lactam antibiotic cephalexin. ... 

Investigation of mass transport limitations for biocatalysts immobilized on heterogeneous supports. 

Researchers in both industry and academia have employed immobilized enzymes as biocatalysts. Immobilization usually involves attaching enzymes to solid supports and packing the supports in a tube through which liquid flows. One application has involved conversion of the lactose in dairy fluids to glucose and galactose, which would permit conversion of the whey produced in cheese manufacture to useful by-products. By averaging over the void spaces between solid particles and the particles themselves, one can obtain an effective rate expression per unit volume of the bed of biocatalyst. The rate expression is... 

FIGURE 44.4 Biocatalysts immobilized by means of (a) crosslinking and (b) co-crosslinking with inert molecules incorporated. 

The choice of biocatalyst, transducer, and method of biocatalyst immobilization all influence sensor design, and many different combinations of microbial biocatalysts and transducers have been investigated. 

The basic principles of bioconversion, bioreactors and biocatalysis are introduced, together with a description of the most important biocatalyst immobilization techniques. The mass transfer phenomena involved in membrane systems are discussed along with some representative configurations of membrane bioreactors, whose behaviour can be described using a simple mathematical approach. For all the aforementioned systems the most significant parameters have been defined to estimate the system performance. 

Steady-state flow reactors, with a constant supply of reactants and continuous removal of products, can be operated as both a continuous stirred-tank bioreactor (CSTB) and as a plug flow bioreactor (PFB). It is possible to have different configurations of the membrane bioreactor where the biocatalyst is immobilized in the fractionated membrane support (Katoh and Yoshida, 2010). In Fig. 1.6 the scheme of a CSMB in which the biocatalyst is immobilized on the surface of the membrane beads is presented. The biocatalyst immobilized in the porous structure of a fractioned membrane can also be operated in CSMB. For example, two configurations are shown in Fig. 1.7 (a) for flat-sheet and (b) for spherical porous structures, respectively. Such structures could also be adopted for PFB, where a bed of membrane support with the immobilized biocatalyst could be utilized, in either a fixed or fluid configuration. 

Biocatalyst immobilized within the membrane polymeric structure. 

Schematic of biocatalyst immobilized onto the membrane surface. Qualitative substrate and product concentration profiles are reported in order to show external mass transfer resistances in the film of thickness 5, when solution flows with velocity U. 

Attachment of biologically active molecules to an insoluble matrix is an essential, and in some instances an indispensable step in the development of biocatalysts. Immobilization allows reuse of enzymes, protects them from harsh external conditions, from microbial contamination, and prolongs their useful lifetime. A large number of existing immobilization techniques reflects the complexity of the biological material and the variety of its applications. Simple inexpensive general techniques, resulting in stable and active enzyme catalysts are yet in demand (25),... 

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