Solid type enzymes

Figure 4. Relative activities of wild type PGII and His223Ala mutated enzyme as a function of pH. Wild type enzyme, solid circles His223Ala mutated enzyme, open circles.

A bioreactor is a vessel in which biochemical transformation of reactants occurs by the action of biological agents such as organisms or in vitro cellular components such as enzymes. This type of reactor is widely used in food and fermentation industries, in waste treatment, and in many biomedical facilities. There are two broad categories of bioreactors fermentation and enzyme (cell-free) reactors. Depending on the process requirements (aerobic, anaerobic, solid state, immobilized), numerous subdivisions of this classification are possible (Moo-Young, 1986). 

A small selection of major reaction types has been studied, but the list is by no means complete. Enzymes, solid acids such as zeolites and Envirocats (see Table 16.3), and new oxidation catalysts are obvious cases where the new technology can bring genuine improvements to chemistry on an industrial scale. Metallocenes are now causing a revolution in polymer production, replacing conventional Ziegler-Natta catalysts for as many as sixty monomers [28]. Further catalyst improvements are still desirable, particularly in the areas of oxidation and aromatic substitution. 

Ion exchange resins, H-Z type solid acids. Enzymes, Solid super acids. Ion exchange membranes... 

Flotation is another method to remove solid (cells) from fermentation broth, using air bubbles to float protein. We may use different kinds of high shear-force devices to make homogeneous solutions for liberating intracellular enzymes. Figure 7.8 shows several types of impeller for homogenisation. 

For most applications, enzymes are purified after isolation from various types of organisms and microorganisms. Unfortunately, for process application, they are then usually quite unstable and highly sensitive to reaction conditions, which results in their short operational hfetimes. Moreover, while used in chemical transformations performed in water, most enzymes operate under homogeneous catalysis conditions and, as a rule, cannot be recovered in the active form from reaction mixtures for reuse. A common approach to overcome these limitations is based on immobilization of enzymes on solid supports. As a result of such an operation, heterogeneous biocatalysts, both for the aqueous and nonaqueous procedures, are obtained. 

There is considerable experimental evidence indicating loss of biological activity of macromolecules such as globular proteins and enzymes at gas-Hquid [57], liquid-solid (Fig. 26) [107] and liquid-liquid [108] interfaces. The extent of inactivation has been shown to be strongly influenced by the prevailing flow field and by, many other factors including the presence and/or absence of additives and contaminants and the type of solid surfaces (Figs. 27 and 28) [107]. 

Among the five different species of Azospirillurn, only A. irakeme shows clearly pectinolytic activity on solid and in liquid medium. Moreover, this species can grow under non-diazotrophic as well as diazotrophic conditions when pectin is the sole carbon source (Khammas and Kaiser, 1991). Khammas and Kaiser (1991) analysed the pectinolytic activity of seven A. irakense isolates, and gave evidence for the presence of two types of pectinolytic enzymes. All strains tested have inducible Ca dependent pectate lyase activity. Six strains, showed also pectin methylesterase activity. So far, none of the corresponding enzymes have been purified. 

When supported complexes are the catalysts, two types of ionic solid were used zeolites and clays. The structures of these solids (microporous and lamellar respectively) help to improve the stability of the complex catalyst under the reaction conditions by preventing the catalytic species from undergoing dimerization or aggregation, both phenomena which are known to be deactivating. In some cases, the pore walls can tune the selectivity of the reaction by steric effects. The strong similarities of zeolites with the protein portion of natural enzymes was emphasized by Herron.20 The protein protects the active site from side reactions, sieves the substrate molecules, and provides a stereochemically demanding void. Metal complexes have been encapsulated in zeolites, successfully mimicking metalloenzymes for oxidation reactions. Two methods of synthesis of such encapsulated/intercalated complexes have been tested, as follows. 

Most of the isotherms reported for the adsorption of enzymes on solids are of the H or L types and are best fitted either by the Langmuir or the Freundlich model (Table 15.3). 

Although not all facets of the reactions in which complexes function as catalysts are fully understood, some of the processes are formulated in terms of a sequence of steps that represent well-known reactions. The actual process may not be identical with the collection of proposed steps, but the steps represent chemistry that is well understood. It is interesting to note that developing kinetic models for reactions of substances that are adsorbed on the surface of a solid catalyst leads to rate laws that have exactly the same form as those that describe reactions of substrates bound to enzymes. In a very general way, some of the catalytic processes involving coordination compounds require the reactant(s) to be bound to the metal by coordinate bonds, so there is some similarity in kinetic behavior of all of these processes. Before the catalytic processes are considered, we will describe some of the types of reactions that constitute the individual steps of the reaction sequences. 

Redox enzymes have been assembled in a monolayer on the solid surface by a potential-assisted self-assembling method as well as a thiol-gold selfassembling method. These enzymes are electronically communicated with the solid substrate through a molecular interface of conducting polymer and a covalently bound mediator. Electron transfer type of enzyme sensors have been fabricated by the self-assembling methods. 

Reactions of cell growth or those using immobilized enzymes are instances of gas-liquid-solid reactions. In principle, accordingly, any of the types of reactors described in Section 8.3 could be employed as fermentors. Mostly, however, mechanically agitated tanks are the type adopted. Aeration supplies additional agitation as well as metabolic need, and moreover sweeps away C02 and noxious byproducts. 

Roth and Breaker1331 used in vitro selection to evolve DNA molecules of the type illustrated in Scheme 4, which self-cleave in the presence of histidine. (This is in principle mimicking a disabled mutant enzyme, of the sort mentioned in the introduction.1101) In this case a pool of 2 x 1013 modified DNAs was attached at the 5 -end, tightly but reversibly, to a solid support The polynucleotide was made up of a randomised sequence of 40 deoxynucleotides (N40 in Scheme 4) flanked by two regions of basepairing complementarity to the sequences on either side of a single upstream RNA linkage (rA). 

As can be concluded from this short description of the factors influencing the overall reaction rate in liquid-solid or gas-solid reactions, the structure of the stationary phase is of significant importance. In order to minimize the transport limitations, different types of supports were developed, which will be discussed in the next section. In addition, the amount of enzyme (operative ligand on the surface of solid phase) as well as its activity determine the reaction rate of an enzyme-catalyzed process. Thus, in the following sections we shall briefly describe different types of chromatographic supports, suited to provide both the high surface area required for high enzyme capacity and the lowest possible internal and external mass transfer resistances. 

Chromatographic fixed-bed reactors consists of a single chromatographic column containing a solid phase on which adsorption and reaction take place. Normally a pulse of reactant is injected into the reactor and, while traveling through the reactor, simultaneous conversion and separation take place (Fig. 3). Since an extensive overview of the models and applications of this type of reactor was presented by Sardin et al. [ 132], only a few recent results will be discussed here. Most of the practical applications have been based on gas-liquid systems, which are not applicable for the enzyme reactions, but a few reactions were also reported in the liquid phase. One of these studies, performed by Mazzotti and co-workers [ 141 ], analyzed the esterification of acetic acid into ethyl acetate according to the reaction ... 

To further extend the utility of structural methods, researchers compare solid state X-ray crystallographic and solution-state NMR structures to define important differences. For instance, the Bertini group has studied the enzyme matrix metalloproteinase 12 (MMP12), in the presence of its inhibitors. Matrix metalloproteinases (MMPs) are involved in extracellular matrix degradation, a fundamental step in tissue remodeling and repair. There are a great variety of enzymes of this type, the one studied here is one of many found in humans. Most MMPs have three domains (1) a prodomain that is removed... 

---