Fluid enzymatic reactions

Process Va.ria.tlons. The conventional techniques for tea manufacture have been replaced in part by newer processing methods adopted for a greater degree of automation and control. These newer methods include withering modification (78), different types of maceration equipment (79), closed systems for fermentation (80), and fluid-bed dryers (81). A thermal process has been described which utilizes decreased time periods for enzymatic reactions but depends on heat treatment at 50—65°C to develop black tea character (82). It is claimed that tannin—protein complex formation is decreased and, therefore, greater tannin extractabiUty is achieved. Tea value is beheved to be increased through use of this process. 

Levels of a number of metabolites as well as a number of enzymes in body fluids are indicative of disease conditions. Many of the enzymatic reactions mentioned above have been used in solution clinical assays as well as in test strips.446,497-508 512-515 Assays for hydrogen peroxide and the enzyme peroxidase using NADH and a tetrazolium salt have been de-scribed.509,5io Assays of exogenous substances (e.g., drugs or their metabolites) also utilize this chemistry. The determination of alcohol using alcohol dehydrogenase is an example.511 As mentioned above, the assay of enzyme levels can also be achieved using tetrazolium salts.516-520... 

This chapter discusses the steps involved in the development and design of a new S02 oxidation catalyst VK69, which was introduced to the market in 1996 by Haldor Topsoe. The strategy and many of the methods are generally applicable to heterogeneous fixed bed catalysts, partly to fluid and slurry bed catalysts, and less relevant for homogeneous catalysts as found in organic synthesis and enzymatic reactions. 

Z. Knez, Supercritical fluids as a solvent for industrial scale enzymatic reactions - yes or no technical lecture invited by DECHEMA for the 2nd COST (Cooperation in Science and Technology - Commission of the European Communities) Workshop on chemistry under extreme or non-classic conditions, Lahnstein, 1995. 

The supercritical fluid extraction of oil seeds has been investigated extensively by several authors [34,98]. Possible applications of supercritical fluids in the edible-oil industry include deacidification, deodorization, and fractionation of crude oils and chemical conversion (like hydrogenation, and enzymatic reactions). 

Dumont, T. Barth, D. Perrut, M. Continuous Synthesis of Ethyl Myristate by Enzymatic Reaction in Supercritical Carbon Dioxide. J. Supercrit. Fluids 1993, 6, 85-89. 

Kamat, S. Critchley, G. Beckman, E. J. Russell, A. J. Biocatalytic Synthesis of Acrylates in Organic Solvents and Supercritical Fluids HI. Does Carbon Dioxide Covalently Modify Enzymes Biotechnol. Bioeng. 1995, 46, 610-620. Kamihira, M. Taniguchi, M. Kobayashi, T. Synthesis of Aspartame Precursors by Enzymatic Reaction in Supercritical Carbon Dioxide. Agric. Biol. Chem. 1987, 51, 3427-3428. 

Amperometric detection was achieved on two patches of C films (formed by CVD of 3,4,9,10-perylenetetracarboxylie dianhydride) on a glass chip. The microchannels were formed using a 23- im-thick photoresist as a spacer. Glucose oxidase and lactate oxidase were immobilized with HRP on the C films via a coated film of osmium PVPD polymer. Simultaneous measurements of glucose and lactate in rat brain cerebrospinal fluid (first perfused with 50 mM veratridine) were achieved. These two films were spatially separated in order to avoid interdiffusion of H202 formed from the two separate enzymatic reactions. Moreover, the two films were preceded by a third C film immobilized with ascorbate oxidase in order to remove ascorbic acid interference [759]. 

In some cases, substrates and enzymes are not soluble in the same solvent. To achieve efficient substrate conversion, a large interface between the immiscible fluids has to be established, by the formation of microemulsions or multiple-phase flow that can be conveniently obtained in microfluidic devices. Until now only a couple of examples are published in which a two-phase flow is used for biocatalysis. Goto and coworkers [431] were first to study an enzymatic reaction in a two-phase flow in a microfluidic device, in which the oxidation ofp-chlorophenol by the enzyme laccase (lignin peroxidase) was analyzed (Scheme 4.106). The surface-active enzyme was solubilized in a succinic acid aqueous buffer and the substrate (p-chlorophenol) was dissolved in isooctane. The transformation ofp-chlorophenol occurred mainly at... 

Enzymatic reactions in non-aqueous solvents are subjected to a wide interest. A particular class of these solvents is the supercritical fluid (1) such as carbon dioxide that has many advantages over classical organic solvents or water no toxicity, no flammability, critical pressure 7.38 Mpa and temperature 31°C, and allowing high mass transfer and diffusion rates. 

The first is the presence of proteolytic activities, which must be inhibited early in the procedure to prevent the degradation of other enzyme proteins. Second, the amount of protein present in these fluids is usually in excess of what an HPLC analytical column can handle without becoming clogged. And finally, these fluids often contain many low molecular weight compounds, either those added as nutrients or those present as a result of cellular metabolism. Since such compounds may resemble either the substrate or product, or both, of the enzymatic reaction under study, their presence in the reaction mixture could interfere with the assay. At the very least, such compounds will pass through the analytical column and appear on a chromatogram, confusing the experimental results. 

In addition to chemicals, biological catalysts such as enzymes can be used to catalyze reactions in SC CO2. Since the first attempt to operate reactions in supercritical fluids published by Randolph et al. [34], various type of enzymes were studied lipase, oxidase, decarboxylase, dehydrogenase, proteinase, etc. [33,35-37]. The effect of different parameters was extensively reported by Ballesteros et al. [35]. Enzyme activity and stability in supercritical conditions as well as the benefits of using supercritical fluids for enzymatic reactions (improved reaction rates, control of selectivity, etc.) have been demonstrated [36]. 

Knez Z and Habubn M. Compressed gases as alternative enzymatic-reaction solvents A short review. J. Supercrit. Fluids 2002 23(1) 29 2. 

Diffusion is an efficient means of materials transport in natural and artificial nanoscale systems and can be readily employed in the study of enzymatic reactions in fluid membrane reactors of static or of changing geometries and morphologies. Other means of transport, e.g. electrophoretic or tension-driven modes are also available. 

Enzymatic catalysis Enzymatic reactions and cell behavior in supercritical fluids [64]... 

Magnesium is the fourth most abundant cation in the body after sodium, potassium, and calcium, and is the second most abundant cation in intracellular fluid after K+ Mg + is needed in many enzymatic reactions, particularly those in which ATP Mg + is a substrate. Magnesium binds to other nucleotide phosphates and to nucleic acids and is required for DNA replication, transcription, and translation. The DNA helix is stabilized by binding... 

Perrut M. Enzymatic reactions and cell behaviour in supercritical fluids. Chem Biochem Eng Q 1995 8 25-30. 

One of the most important assumptions in MM kinetics is that the reaction in question wiU proceed in a three-dimensional vessel filled with a well-stirred fluid that obeys Pick s law for diffusion. This is rarely the case in a living cell, where many reactions are localized to membranes (two dimensions) or to small regions somewhere within the cell, creating an effectively one-dimensional environment with little or no diffusion. To circumvent this limitation, fractal kinetics have been developed which allow for the approximation of enzymatic reaction velocities in vivo [7]. Fractal kinetics can utilize MM-type kinetic constants to create a model of events in a spatially restricted environment. Briefly, as the dimensionality of a reaction is reduced from three dimensions to one, the kinetic order of a bimolec-ular reaction, for example, increases from 2 in a three-dimensional case, to 2.46 in a two-dimensional environment (e.g., membrane), to 3 in a one-dimensional channel, up to 50 for the case where fractal dimensions are less than 1. In simple terms, the kinetic order is the sum of all stoichiometric coefficients of the reactants in a balanced chemical reaction equation. Rearranging the familiar equation for MM kinetics... 

In pioneering research by Hailing and co-workers, it was demonstrated that the activity of water is a more representative and useful parameter than water concentration for describing enzymatic rates in nonaqueous enzymology. Water activity, or is defined as the fugacity of water contained in a mixture divided by the fugacity of pure water at the mixture s temperature. For a typical nonaqueous enzymatic reaction operated in a closed system, the medium will consist of a solvent (or fluid) phase, an enzyme-contaiifing solid phase, and air headspace above the solvent. As a first approximation, the water transport between the three phases is assumed to be at thermodynamic equilibrium. For such a situation, can be defined in terms of the air headspace properties ... 

Enzymatic reactions have been monitored by several procedures. In the case of solid-phase enzymes, analysis is best achieved by periodically withdrawing small aliquots of fluid-phase reaction medium, after solid-fluid separation has occurred via gravity settling (e.g., disabling the agitator in batch reactors), filtration, or centrifugation. The aliquot can then be analyzed via chromatography or spectroscopy. Water content... 

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