Enzymatic Reactor Configurations

Highly soluble (dyes, Retention of the Enzyme recovery by Monophasic reactor Basheer et al. (1993) Pasta et al. 

The design of enzymatic reactors for the degradation of highly soluble compounds may consider the use of a semi permeable membrane for the separation of the enzyme and products or substrates (enzyme membrane reactors) or the immobilization of the enzyme in a support (immobilized enzyme reactors). 

The system is provided with a membrane of suitable molecular cut-off, which acts as a selective barrier for the retention of the enzyme. Permeable substrates and products are taken out from the reaction mixture by the action of a gradient (chemical potential, pressure) through the membrane. Based on the combination of membranes and enzyme reactors two main configurations are considered, as shown in Fig. 6.6.2. In the first configuration, the enzyme may be immobilized by covalent binding between an activated group of the membrane and a functional group of the protein 

References about immobilized enzyme reactors for environmental purposes are scarce (Katchalski-Katzir and Kraemer 2000). The concept is based on the immobilization of the enzyme onto a support by covalent binding or ionic interaction. The feasibility of the immobilized enzyme reactors is determined by the following requirements i) the specific activity of the derivative (units of enzyme per g of support) should be as high as possible ii) the support or membrane should be applied with a secondary function, such as the separation of substrates or products and iii) the support should have good mechanical resistance and minimum interaction with the substrates or products. Additionally, the immobilization process should be simple and inexpensive. 

Different reactor configurations are proposed for the use of immobilized enzymes (Fig. 6.6.3) A) stirred tank reactors B) fixed bed reactors and C) fluidized bed reactors. The selection of the best option depends on the type of support and reaction kinetics. However, very often the activated supports do not present adequate characteristics for the performance of a fixed or fluidized bed reactor and, therefore, the development of stirred tank reactors based on immobilized enzyme or even, sequential stirred tank reactors appear as the more feasible options. 

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Aim of this work was to optimise enzymatic depolymerization of pectins to valuable oligomers using commercial mixtures of pectolytic enzymes. Results of experiments in continuous and batch reactor configurations are presented which give some preliminary indications helpful to process optimisation. The use of continuous reactors equipped with ultrafiltration membranes, which assure removal of the reaction products, allows to identify possible operation policy for the improvement of the reaction yield. 

The discontinuous stirred tank reactor represents one of the most traditional reactor configurations for enzymatic reactions. It consists of a stirred tank where the enzyme, substrates, and cofactors are added at the beginning of the operation with no inlet and/or outlet stream during the reaction time. This type of reactor is usually considered to present an ideal hydrodynamic behavior therefore, the reactor is supposed to be completely mixed and the concentration of all... 

This chapter first explains enzyme nomenclature, describes enzymatic, supercritical reactor configurations, and gives a compilation of published experimental results. The- most important topics concerning enzymatic reactions in SCFs are then covered. These are factors affecting enzyme stability, the role of water in enzymatic catalysis, and the effect of pressure on reaction rates. Studies on mass transfer effects are also reviewed as are factors that have an effect on reaction selectivities. Finally, a rough cost calculation for a hypothetical industrial process is given. 

Table 6.6.3 Possible Configurations of Enzymatic Reactors Treating Highly or Poorly Soluble Compounds...

The process is configured as a series of three stirred-tank reactors with the substrate 3-cyanopyridine continuously fed at 10-20 wt.% concentration and the biocatalyst flowing countercurrently. Enzymatic hydrolysis yields the desired nicotinamide at > 99.3% selectivity, in contrast to the chemical alkaline hydrolysis process which results in about 3-5% nicotinic acid, an undesirable by-product because it causes diarrhea in farm animals (instead of supporting growth for animal feed supplements, see Chapter 6, Section 6.4). Thus, the enzymatic process competes well with the chemical hydrolysis. 

In recent years, membrane bioreactors, bioreactors combined with membrane separation unit have established themselves as an alternative configuration for traditional bioreactors. The important advantages offered by membrane bioreactors are the several different types of membrane modules, membrane structures, materials commercially available. Membrane bioreactors seem particularly suited to carry out complex enzymatic/microbial reactions and/or to separate, in situ, the product in order to increase the reaction efficiency. The membrane bioreactor is a new generation of the biochemical/chemical reactors that offer a wide variety of applications for producing new chemical compounds, for treatment of wastewater, and so on. 

Immobilized /3-glucosidase served for enzymatically catalyzed hydrolysis of benzene metabolites in urine. End analysis of phenol was by RP-HPLC with ELD at 0.85 V vi. Ag/AgCl electrode. ELD avoids interference from other componnds present in urine. LOD was 10 p.gL" (20 p,L injection, 0.2 ng), with RSD 1.16% and 3.38% for 1.2 ng and 2.0 ng, respectively. A stndy was carried out of two FIA systems for enzymatically catalyzed determination of dopamine (10a). Thus, a combination of a packed bed reactor containing immobilized tyrosinase followed by photometric detection was compared with ELD based on a graphite electrode with its surface covered by immobilized tyrosinase. The former configuration was linear up to 0.75 mM while the latter reached 1 mM. LC separation and post-column detection with the bioelectrode was applied to analysis of spiked serum samples. ... 

As was described above in a number of MBR processes the membrane, in addition to performing the separation functions previously discussed, also acts as a host for the biocatalysts (whole cells or enzymes) which are immobilized in the membrane s pore structure. Concerns with such MBR configurations include membrane biofouling, mass transport limitations and biocatalyst activity loss and denaturation. In the two sections that follow we discuss further some of the key aspects of MBR for biochemical synthesis. We classify these reactors into two types, namely whole-cell and enzymatic MBR. 

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.

Fig. 6.6.2 Schemritic diagrams of different configurations of enzymatic membrane reactors (a) stirred tank reactor with enzyme immobilized or retained by a membrane (b) stirred tank reactor coupled to ultrafiltration membrane...

Different sample pretreatment operations include dilution, membrane-extraction (gas diffusion, dialysis), liquid-phase extraction techniques (liquid/liquid extraction, liquid-phase microextraction, single-drop microextraction) and solid reactors and packed columns aiming to facilitate online chemical derivatization, chromatographic separation of target species, removal of interfering matrix compounds, enzymatic assays, or determination of trace levels of analyte via sorptive preconcentration procedures (Marshall et al., 2003 Economou, 2005 Miro and Hansen, 2006 Theodoridis et al., 2007 McKelvie, 2008 Ruzicka, 2014). In this context, BIA and the LOV configurations are particularly useful. Acid-base titrations can also be automated using simple SIA manifolds and potentiometric (van Staden et al., 2002) or photometric (Kozak et al., 2011) detection. Typically, a zone of the sample to be titrated is sandwiched between two zones of titrant by aspiration. In the case of photometric detection, an additional zone of a suitable pH-sensitive colored indicator is aspirated. The stacked zones are delivered to the detector and the width of the peaks is monitored and related to the pH of the solution. 

The simultaneous determination of sulfite and phosphate in wine was proposed by Yao et al. (1994), where a FI A system was coupled with amperometric detection. In this approach two different reactors with immobilized sulfite oxidase and co-immobilized purine nucleoside phosphorylase-xanthine oxidase were incorporated in a parallel configuration. The enzymatically generated hydrogen peroxide was selectively detected on a poly(l,2-diaminobenzene)-coated platinum electrode. Because of the different residence times at each channel, two different peaks could be obtained, the first corresponding to sulfite and the second to phosphate. Using this system, the analytes could be simultaneously analyzed with a linear range of 1 x 10 -2 x 10 M (sulfite) and 2 x 10 -5 x 10 M (phosphate) and a sample throughput of 30 Mb... 

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