Enzymes reactor conditions

Physiological optimization of enzyme synthesis by variation of the culture parameters is usually required to enhance the catalytic activity of whole-cell biocatalysts to such a level that it can be apphed in a biocatalytic process. In addition, physiological conditions can influence the selectivity of the reaction, since enzymes with opposite selectivities can be differentially expressed. In some cases, genetic engineering is required to obtain biocatalysts with a desired selectivity that does not consume the product of choice (see 5.3.5). Alternatively, one may choose to isolate the desired activity from the culture in order to use the biocatalyst in an enzyme reactor. 

Figure 2. The effect of chemical modification of lysyl E-amino groups on the amount of bound enzyme at steady-state reactor conditions...

Form of enzyme and concentration of substrate influence the yield and type of lOS produced, however another study by Yun et al. [277] used whole cells of Escherichia coli HB101 whereby the gene for an endo-inulinase from Pseudomonas sp was expressed. In a batch reactor the production of lOS using the recombinant enzyme was compared to that of the native and there were differences in the DP distribution of the products formed although the yield of lOS was comparable. The native form produced less inulobiose and more DP4 and > DP4 products compared to the recombinant. Additionally Yun et al. [277] studied the effect of different reactor conditions batch processing versus continuous using immobilized recombinant cells but no differences in product composition were observed. 

Transport of solutes through the LM occurs by either passive transport or by carrier-facilitated transport. Phenol, for example, is soluble in both phases, and treatment of an aqueous phenol solution with an emulsion results in a lowering of the external concentration of phenol as it passively diffuses through the hydrocarbon (HC) layer and into the internal aqueous phase. Equilibrium is reached when the concentrations of phenol in both aqueous solutions are equal (assuming no other conditions are present which would alter the distribution between the aqueous and HC phases). One way to alter this equilibrium is to trap phenol inside with a sodium hydroxide solution. Phenol ionizes at high pH, and the phenolate ion cannot permeate a HC layer trace amounts of phenol have been completely removed from wastewaters by this system (10, 11). This exclusion of charged molecules by the aliphatic hydrocarbon LM layer is desirable in some applications, but to employ LM enzyme reactors and/or separation systems with amino acids, it is necessary to incorporate carriers into the HC phase. 

The purpose of studies on enzyme properties is to select favorable reaction conditions for the investigation of enzyme kinetics. The choice of assay conditions has to be performed very carefully, and it has to be proven that the assay conditions are as close as possible to the reactor conditions of the final process. This aspect cannot be stressed too much ... 

The overall reaction rate not only determines the necessary reaction time to reach the desired conversion (see Fig. 7-23) but also the enzyme and cofactor consumption, both being strongly influenced by the reactor conditions. 

The enzyme ratio represents the ratio of the initial volumetric activities of the enzymes (dimensions U mL 1). For process conditions, enzyme activity under the actual steady-state reactor conditions is significant, and differs from initial rate conditions as determined by enzyme kinetics. Therefore, the optimum enzyme ratio, implying maximum conversion within minimum residence time, is not 0.5. 

Another concern with packed-bed reactors is reproducibility. There are two parts to reproducibility can multiple packed-bed reactors be made reproducibly and what is the stability of the reactor The first question concerning reproducibility can be answered as follows. The art of packing a column is at times difficult and each individual analyst will have different success. However, all packed-bed reactors can be calibrated in the FIA system. A variation in the performance level between reactors of less than 10% is acceptable. The second reproducibility question concerns the loss in reactivity due to reagent degradation or saturation of the reactive sites. In the ideal case the reactivity of the column should not change with respect to time, thereby producing a reproducible signal for the same concentration of analyte. This is a more difficult problem. Ideally, the analyst should find the reactor conditions that will minimize loss of activity. In practice, especially with enzymes, this condition will not be met. Frequent calibrations will be needed to insure the most accurate results. 

Enzyme—1.0 FP activity Substrate—SF-HM (76% < 53/x)—v0% pH— 4.05-5.2 Saccharification temperature—50°C. Dilution rate in continuous phase—0.025"J hr. Reactor conditions 4.0 liter glass stirred tank reactor Feed temperature—1°-2°C. 

Substrates and the SCF are independently pumped together to obtain a single-phase mixture. The mixture is then led through the enzyme reactor. The reactor may be either a tube packed with enzyme or a continuously stirred tank (CSTR). The concentrations, reaction conditions, flow rates, and residence times can be set independently. HPLC pumps are used for pumping the substrates into the SCF, provided that the viscosity of the liquid is low [13, 15]. The SCF and substrates are equilibrated in a separate vessel prior to leading the mixture to the enzyme reactor [16]. Pulse-... 

Mathematical models, especially when coupled with computer techniques, are a very effective tool in searching for optimal operating conditions in the design, operation and control of enzyme reactors. The study of a reliable model for the enzyme reaction system is of significant importance for the industrial application of the biocatalyst. The model has to be effective in a wide range of values of the process variables. 

Based on the optimization of the conditions attained in discontinuous experiments (Mielgo et al. 2003), the degradation of the dye was performed in a continuous enzyme reactor (Ldpez et al. 2004). Different experiments were planned to maximize process efficiency, defined as the ratio between degradation rate and enzymatic consumption. 

The addition of a second immiscible phase for the enzymatic degradation of poorly-soluble compounds provides several advantages, such as a simpler operation, mainly due to the easy recovery of the solvent depleted of substrate and its reuse in subsequent operations. Mass transfer could be considered a priori as a limitation for this system and to be the determinant of lower efficiencies. However, the selection of the appropriate solvent, as well as the determination of the adequate conditions which lead to the maximum efficiency allowed us to obtain unprecedented degradation rates in enzyme reactors. 

There are other published resolutions of 4-hydroxyphenylglycine, one of which involves the enzyme-catalysed hydrolysis of the ethyl ester (Scheme 6.9). Since the substrate is fully blocked at the amino and the carboxylate functions, it is scarcely soluble in water and must be dissolved in an organic solvent. The specific hydrolysis of the ester catalysed by an enzyme in an aqueous phase in contact with the organic solvent will yield a water-soluble carboxylic acid which transfers to the aqueous phase containing the enzyme. The conditions of the reaction therefore effect both hydrolysis of the ester and facile separation of the product. In fact, the enzyme is immobilized on a hydrophilic membrane at the interface between the two immiscible phases. The membrane reactor (Figure 6.2) comprises a large bundle of hollow fibres, each having an external diameter of about... 

The connection of biochemical reaction with electrochemical detection sometimes causes difficulties, for example if the conditions of the biochemical reaction and detector function are different [317, 318]. In such cases it is advantageous to carry out the biochemical reaction separately and to determine the reaction product after changed conditions [319]. Such an enzyme reactor electrode enables a large choice of detection methods, kinds and forms of applied biologically active materials, number of analyses provided per hour and possibilities of automation. Enzyme reactor electrodes are therefore used not only in the above characterized difficult cases, but are often preferred in automated methods of analysis. 

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