Immobilization of the biocatalyst

A procedure for immobilization of a P. stutzeri UP-1 strain using sodium alginate was reported [133], This strain does not perform sulfur-specific desulfurization, but degrades DBT via the Kodama pathway. Nevertheless, the report discussed immobilization of the biocatalyst cells in alginate beads with successful biocatalyst recovery and regeneration for a period of 600 h. However, the immobilized biocatalyst did decrease in specific activity, although the extent of loss was not discussed. The biocatalyst was separated after every 100 h of treatment, washed with saline and a boric acid solution and reused in subsequent experiment. The non-immobilized cells were shown to loose activity gradually with complete loss of activity after four repeat runs of 20 hour each. The report does not mention any control runs, which leaves the question of DBT disappearance via adsorption on immobilized beads unanswered and likewise the claim of a better immobilized biocatalyst. 

In Figure 10.1 the time course of thermodynamically and kinetically controlled processes catalysed by biocatalysts are compared. The product yield at the maximum or end point is influenced by pH, temperature, ionic strength, and the solubility of the product. In the kinetically controlled process (but not in the thermodynamically controlled process) the maximum yield also depends on the properties of the enzyme (see next sections). In both processes the enzyme properties determine the time required to reach the desired end point. The conditions under which maximum product yields are obtained do not generally coincide with the conditions where the enzyme has its optimal kinetic properties or stability. The primary objective is to obtain maximum yields. For this aim it is not sufficient to know the kinetic properties of the enzyme as functions of various parameters. It is also necessary to know how the thermodynamically or the kinetically controlled maximum is influenced by pH, temperature and ionic strength, and how this may be influenced by the immobilization of the biocatalysts on different supports. 

A PPy/PQQ modified GC electrode was used for amperometric detection of V-type nerve agent decomposition products. The electropolymerization of pyrrole was efficiently used for immobilization of the biocatalyst, PQQ. The introduction of CaCl2 as a supporting electrolyte during electrodeposition significantly improves the response of the sensor to DMAET and DEAET. Amperometric studies targeted to detection of DMAET and DEAET by PPy/PQQ electrode were performed at a constant potential set at 0.25 V, and the electrode characteristics such as sensitivity and the analyte detection limit were determined. 

Immobilization of biocatalysts is one method for attempting to increase both current density and stability. However, this strategy can backfire on the researcher. In theory, an en me immobilized on the surface of an electrode surface has lower degrees of freedom and is therefore less likely to denature. The immobilization materials can provide a stabilizing chemical microenvironment for the enzyme. Immobilization can also pre-concentrate biocatalysts at the electrode surface for minimizing electron transport inefficiencies. However, if the biocatalysts are not properly immobilized, the biocatalysts can denature on the electrode surface during immobilization, which results in a decrease in specific activity of the enzyme resulting in lower current densities. Therefore, proper immobilization of the biocatalysts is critical to the properties of the bioelectrode. 

Changing the E value requires a change of the reaction system such as the solvent, the water activity, and immobilization of the biocatalyst. For instance, it has been reported that lipase immobilized on a hydrophobic carrier makes the E value of C. rugosa lipase less sensitive to changes in water activity than the crude enzyme [66]. Other important parameters influencing enantioselectivity are the temperature, the lipase and the lipase formulation, and structural differences in the substrate molecule. Recently, site-directed mutagenesis has attracted increased attention as a tool for altering lipase enantioselectivity [135]. 

The above two processes employ isolated enzymes - penicillin G acylase and thermolysin, respectively - and the key to their success was an efficient production of the enzyme. In the past this was often an insurmountable obstacle to commercialization, but the advent of recombinant DNA technology has changed this situation dramatically. Using this workhorse of modern biotechnology most enzymes can be expressed in a suitable microbial host, which enables their efficient production. As with chemical catalysts another key to success often is the development of a suitable immobilization method, which allows for efficient recovery and recycling of the biocatalyst. 

The development of biphasic media requires a knowledge of general rules based on observation. The choice of the biocatalyst and the organic solvent is very important. Estimation of the biocatalyst tolerance to an organic solvent is based on various indicators, described later in this chapter. Biocatalysts are also sensitive to the process of the liquid-liquid interface. They can be used in two different forms free, soluble or immobilized. 

Several kinds of states in which enzymes may be used for various reactions in aqueous-organic biphasic systems have been developed in previous work (Table 2). In biphasic media, the biocatalyst is easily recovered after the reaction then it is not always necessary to be immobilized. Nevertheless, the immobilization can confer important properties, such as improved stability of biocatalyst. Furthermore, protection of the biocatalyst against a damaging turbulent environment can also play a role. 

Biological catalysts in the form of enzymes, cells, organelles, or synzymes that are tethered to a fixed bed, polymer, or other insoluble carrier or entrapped by a semi-impermeable membrane . Immobilization often confers added stability, permits reuse of the biocatalyst, and allows the development of flow reactors. The mode of immobilization may produce distinct populations of biocatalyst, each exhibiting different activities within the same sample. The study of immobilized enzymes can also provide insights into the chemical basis of enzyme latency, a well-known phenomenon characterized by the limited availability of active enzyme as a consequence of immobilization and/or encapsulization. 

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... 

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. 

As an added benefit, the immobilization resulted in an enhanced half-life of the biocatalyst at elevated temperatures and during storage [12], and thus in an overall improvement of technical applicability. With the entrapped ADH, (Rj-phenylethanol was synthesized from acetophenone with a productivity of... 

Mode of immobilization. Immobilization can be effected either chemically, by covalent bonding of the biocatalyst on a surface (Figure 5.6, option 1), by adsorption, or by ionic interactions between catalyst and surface (option 2), as well as by cross-linking of biocatalyst molecules for the purpose of enlargement (option 3), or physically by encapsulation in matrices or by embedding in a membrane (option 4). 

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. 

One of the greatest hurdles for the application of biocatalysis is the need to operate processes under conditions that can differ dramatically from those in which the enzymes evolved. Many techniques are used in order to preserve catalytic activity and minimize the costs associated with the biocatalyst. In cases where the cost of the biocatalyst is a concern, an enzyme might be immobilized and used in a packed column or a fluidized bed reactor so as to enable reuse. Here also the enzyme must be stable for extended periods and may even be used under nonaqueous conditions and elevated temperatures. Recombinant technology has revolutionized the applications of biocat-... 

Form of the biocatalyst (whole cells, soluble or immobilized enzymes)... 

Another favorable aspect of stirred batch reactors is the fact that they are compatible with most forms of a biocatalyst. The biocatalyst may be soluble, immobilized, or a whole-cell preparation in the latter case a bioconversion might be performed in the same vessel used to culture the organism. Recovery of the biocatalyst is sometimes possible, typically when the enzyme is immobilized or confined within a semi-permeable membrane. The latter configuration is often referred to as a membrane reactor. An example is the hollow fiber reactor where enzymes or whole cells are partitioned within permeable fibers that allow the passage of substrates and products but retain the catalyst. A hollow-fiber reactor can be operated in conjunction with the stirred tank and operated in batch or... 

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