Enzyme bioreactors

A second area of research involves reactors composed of enzymes immobilized to solid supports. Such systems offer a highly specific means of removing undesired substances from the bloodstream. By immobilizing the enzyme, the possibility of inducing an immune response is minimized. Three examples of using immobilized enzymes are now discussed. 

In developing a reactor such as the one just described, it is important to understand important design parameters, such as the radial distribution of the enzyme within the catalyst particles, the kinetics of heparin degradation catalyzed by immobilized heparinase, the flow properties in the reactor, and the effect of in vivo factors such as blood proteins which bind to the substrate. These parameters and how they can be evaluated are now discussed. 

The heparin degradation rate at any radial position inside the catalyst particle is proportional to the bound heparinase concentration at that position. If the immobilized enzyme concentration is not uniform, the conventional analysis of simultaneous diffusion and reaction within a porous catalytic particle must be modified. The reaction rate within the catalyst particle will have an explicit radial dependence introduced via the enzyme concentration, as well as a dependence on the substrate concentration. 

The first step in characterizing the heparinase binding rate to the catalyst particles is to establish experimental conditions where neither enzyme denaturation or external mass transfer are important. This can be accomplished by controlling the duration of immobilization, the mixing rate, and the catalyst particle size. In the absence of diffusional limitations and enzyme denaturation effects, the disappearance of enzymatic activity from the bulk phase equals the rate at which the enzyme binds to the catalyst particle. The molar conservation equation for heparinase in the bulk phase is given by 

The binding kinetics were studied as a function of heparinase concentration at two different cyanate ester concentrations. These cyanate ester groups were present in excess, and this remained constant throughout the experiment. At both ester levels, the disappearance of heparinase from the fluid phase was first-order and the binding constant depended on the cyanate ester concentration (48). 

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This chapter is not intended to serve as a comprehensive review in drug metabolite biosynthesis rather, we will focus on practical considerations for metabolite synthesis at small to medium scale with three bioreactor systems mammalian bioreactors, microbial bioreactors and recombinant enzyme bioreactors. 

For application of protein-immobilized porous materials to sensor fields, use of an electroactive substance as the framework material is important. DeLouise and Miller demonstrated the immobilization of glutathione-S-transferase in electrochemically etched porous silicon films [134], which are attractive materials for the construction of biosensors and may also have utility for the production of immobilized enzyme bioreactors. Not limited to this case, practical applications of nanohybrids from biomolecules and mesoporous materials have been paid much attention. Examples of the application of such hybrids are summarized in a later section of this chapter. 

Fig. 7.n SEM images of the 3D macroporous functionalized macroporous zeolitic membrane zeolitic membrane used as a support for enzyme bioreactor was prepared via the LbL electrostatic immobilization by the LbL procedure. Images assembly of PEs and enzyme (catalase) on the 3D (A-D) are cross-sections of the membrane at macroporous membrane. (Reprinted from [59] different magnifications. A biomacromolecule- with permission of Wiley-VCH). 

An alternative method for coupling enzymes is to employ a mixed enzyme bioreactor Pezzotti and Therisod [60] reported a bienzymatic system in which alcohol oxidase and peroxidase were coupled to effect the enantioselective oxidation of the sulfide thioanisole (Scheme 3.4). Here, the peroxidase from Coprinus dnereus was mixed with a crude extract of Pichia pastoriz alcohol oxidase and the two enzyme mixture was successfully used to convert gram quantities of thioanisole enantiose-lectively to S-methyl-phenyl-sulfoxide with an enantiomeric excess of 75%. 

Isono Y, Nakajima M (2000) Membrane phase separation of aqueous/alcohol biphase mixture and its application for enzyme bioreactor. Prog Biotechnol 2000 63-68... 

Another promising area for adaptation of enzyme bioreactor technology is that of lipid modification. Several examples are a) the interesterification of triacylglycerols to change their composition b) limited lipolysis for production of flavors and c) conversion of cholesterol to forms that are not absorbed. The potential stabilization of enzymes to the presence of organic solvents would provide a definite advantage to enzyme bioreactor technology for the modification of lipid molecules. 

In our view, genetic engineering of future enzymes for industrial uses should consider not only their catalytic properties, but also their potential for isolation and immobilization. Designing enzymes to allow selective, high affinity immobilization by adsorption on a relatively inexpensive matrix should greatly increase the attractiveness of enzyme bioreactor processes. 

The diversity of compounds that can be analyzed by HPLC currently preclude the use of any one HPLC/MS technique to specifically detect trace quantities. Thermospray is one of the most popular HPLC/MS techniques due to its ability to ionize nonvolatile and thermally labile compounds with minimal compromises on a HPLC separation or MS operation. Ion evaporation ionization (no filament or discharge) is suitable for numerous compounds, but often results in spectra with insufficient structural information (i.e. one ion spectra), with sensitivities varying drastically between compound classes. In cases where thermospray specificity or sensitivity is not sufficient, complementary HPLC/MS approaches need to be employed. The use of thermospray HPLC/MS and complementary techniques (i.e. immobilized enzyme bioreactors, chemical degradation and particle beam HPLC/MS) have been evaluated for the specific analysis of three major classes of compounds--peptides, pharmaceuticals, and pesticides. 

The activity of the immobilized enzyme bioreactor plays an important role in the quality of the spectra. Immobilized enzyme activity is dependent on pH, temperature, solvents and buffers used for the hydrolysis. Extremes in any one condition can irreversibly destroy the bioreactor activity. Hydrolysis at conditions far from optimal can lead to no-hydrolysis or partial hydrolysis of a peptide, providing limited information on the peptide. In order to successfully employ an immobilized enzyme column on-line with HPLC/thermospray MS, organic modifiers must be kept minimal (less than 30-50%), pH must be between 6.5 and 8.5 and buffer (ammonium acetate) concentration around 0.05-0.1M [12]. 

One can also envision applications involving the use of encapsulated or entrapped enzymes in bioreactors for therapeutic applications involving detoxification of deleterious substances or correction of metabolic deficiencies. In these applications, the enzymes could be contained within artificial cells [e.g., modified red blood cells (erythrocytes) or liposomes]. Liang, Li, and Yang have reviewed biomedical applications of immobilized enzyme bioreactors. 

The rising need for new separation processes for the biotechnology industry and the increasing attention towards development of new industrial eruyme processes demonstrate a potential for the use of liquid membranes (LMs). This technique is particularly appropriate for multiple enzyme / cofactor systems since any number of enzymes as well as other molecules can be coencapsulated. This paper focuses on the application of LMs for enzyme encapsulation. The formulation and properties of LMs are first introduced for those unfamiliar with the technique. Special attention is paid to carrier-facilitated transport of amino acids in LMs, since this is a central feature involved in the operation of many LM encapsulated enzyme bioreactor systems. Current work in this laboratory with a tyrosinase/ ascorbate system for isolation of reactive intermediate oxidation products related to L-DOPA is discussed. A brief review of previous LM enzyme systems and reactor configurations is included for reference. 

Zhu D, Zhu Y, Cai H, Gao G, Gao C, Li B. Method for treating leather waste to produce coUagen with high added value by using enzyme bioreactor and membrane separator (Shenzhen Xianke Environment Protection Co., Ltd, People s Republic of China). Patent Priority CAN 145 194650 AN 2006 754551 (in Chinese). 

Tl. Tabata, M., and Murachi, T., A chemiluminometric method for the determination of urea in serum using a three enzyme bioreactor. J. Biolumin. Chemilumin. 12, 63-67 (1988). 

Isono Y, Nakajima M (2000) Membrane phase separation of aqueous/alcohol biphase mixture and its application for enzyme bioreactor. Prog Biotechnol 16 63-68 Jaspers CJ, Jimenez G, Penninckx MJ (1994) Evidence for a role of manganese peroxidase in the decolorization of Kraft pulp bleach plant effluent by Phanerochaete chrysosporium effects of initial culture conditions on enzyme production. J Biotechnol 37(3) 229-234 Johannes C, Majeherezyk A, Huttermaim A (1996) Degradation of anthracene by laccase ofTram-etes versicolor in the presence of different mediator compounds. Appl Microbiol Biotechnol 46(3) 313-317... 

In enzyme bioreactors (EBRs), in which the enzyme is used in homogeneous conditions and no membrane separation occurs, productivity can be evaluated as ... 

Other interesting appUcations regard (S)-Naproxen through biphasic EMRs. In these cases, the presence of the emulsion combined with the enzyme bioreactor improves the catalytic activity and enantiose-lectivity of the immobilized enzyme, along with the mass transfer of the hydrophobic reagent (Naproxen esters and triglycerides) note that the immobilization procedure preserves enzyme stability without altering its enantioselectivity. 

Beeause F is the volume processed in time f in a continuous flow bioreactor and Vl is the corresponding volume in a batch reactor, a comparison of Eqs. (11) and (13) shows that batch and plug flow (i.e., packed bed) bioreactors containing the same amount of enzyme will achieve equal conversions in a given time. This is a general conclusion, irrespective of the reaction kinetics. A continues flow packed bed enzyme bioreactor may be advantageous relative to batch reactor, as the unproductive time for batch preparation could be eliminated in the continuous flow unit. However, the batch reactor may have other important advantages such as the ease of pH control in a well-mixed device. 

Kang, Y.W., Kang, C., Hong, J.S., and Yim, S.E. (2001) Optimization of the mediated electrocatalytic reduction of NAD by cydic voltammetry and construction of electrodiemically driven enzyme bioreactor. Biotechnol. Lett., 23,... 

Derksen, J.T.P., Muuse, B.G., Cuperus, RP, Van Gelder, W.M.J., 1992. New seed oils for oleochemical industry evaluation and enzyme-bioreactor mediated processing. Ind. Crops Prod. 1,133-139. 

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