Scale 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. 

Synthesis of DHAP. Both RAMA and Fuc-l-P aldolase require DHAP as the nucleophilic component. Although DHAP is commercially available, it is too expensive for synthetic use, and must be synthesized for preparative-scale enzymatic reactions. DHAP has been prepared via three major routes enzymatically from FDP using a combination of RAMA and triose isomerase (TIM, E.C. 5.3.1.1) (25), chemically, by phosphorylation of dihydroxyacetone dimer (26), and enzymatically by phosphorylation of dihydroxy acetone catalyzed by glycerokinase (Scheme 8) (25). 

The characteristic times on which catalytic events occur vary more or less in parallel with the different length scales discussed above. The activation and breaking of a chemical bond inside a molecule occurs in the picosecond regime, completion of an entire reaction cycle from complexation between catalyst and reactants through separation from the product may take anywhere between microseconds for the fastest enzymatic reactions to minutes for complicated reactions on surfaces. On the mesoscopic level, diffusion in and outside pores, and through shaped catalyst particles may take between seconds and minutes, and the residence times of molecules inside entire reactors may be from seconds to, effectively, infinity if the reactants end up in unwanted byproducts such as coke, which stay on the catalyst. 

Enzymatic degradation of pectin can be satisfactory performed in UF-membrane reactors which have been proved to be helpful tool for laboratory scale investigations. Reaction products can be continuously recovered in a sequence of filtration stages. The obtained product distribution depends on the enzyme to substrate ratio, which affects particularly the... 

There have been many papers reporting studies on the influence of structure and conditions of the medium. Specifically, the kinetic behavior of enzymatic reactions in two-phase media was probed [7,25,27,63]. The reaction localization and the interaction between mass transfer and metabolism in compartmentalized media are interesting phenomena. Their study in the laboratory are useful for optimizing the operating conditions of bioreactors in a preparative scale. In addition, they also help to understand better the behavior of enzymatic systems in vivo. 

For the quantitative description of the metabolic state of a cell, and likewise which is of particular interest within this review as input for metabolic models, experimental information about the level of metabolites is pivotal. Over the last decades, a variety of experimental methods for metabolite quantification have been developed, each with specific scopes and limits. While some methods aim at an exact quantification of single metabolites, other methods aim to capture relative levels of as many metabolites as possible. However, before providing an overview about the different methods for metabolite measurements, it is essential to recall that the time scales of metabolism are very fast Accordingly, for invasive methods samples have to be taken quickly and metabolism has to be stopped, usually by quick-freezing, for example, in liquid nitrogen. Subsequently, all further processing has to be performed in a way that prevents enzymatic reactions to proceed, either by separating enzymes and metabolites or by suspension in a nonpolar solvent. 

The main drawback of the DHAP-dependent aldolases is their strict specificity for the donor substrate. Apart from the scope limitation that this fact represents, DHAP is expensive to be used stoichiometrically in high-scale synthesis, and labile at neutral and basic pH, and therefore its effective concentration decreases over time in enzymatic reaction media, hindering the overall yield of the aldol reaction. In addition, due to the presence of a phosphate group in both DHAP and the... 

Membrane reactors have been investigated since the 1970s 11). Although membranes can have several functions in a reactor, the most obvious is the separation of reaction components. Initially, the focus has been mainly on polymeric membranes applied in enzymatic reactions, and ultrafiltration of enzymes is commercially applied on a large scale for the synthesis of fine chemicals (e.g., L-methionine) 12). Membrane materials have been improved significantly over those applied initially, and nanofiltration membranes suitable to retain relatively small compounds are now available commercially (e.g., mass cut-off of 400—750 Da). 

Sheldon, R.A. (1996) Large-scale enzymatic conversions in non-aqueous media. Enzymatic Reactions in Organic Media, A.M.P.Koskinen and A.M.Klibanov. London Chapman Hall. 

The enzyme recLBADH is the first catalyst that has been found to allow the highly regio- and enantioselective synthesis of 5-hydroxy-P-keto esters by reduction of the respective diketo esters. This enzymatic reaction is of enormous preparative value. The substrates are readily available by acylation of P-keto ester bisenolates and the reaction only requires a simple batch technique which is easy to scale up. Reduction of the chlorinated compound la has been performed routinely on a 75 g scale in our laboratory (8 L fed batch), yielding (S)-2a in an isolated yield of 84% [10]. 

In order to study the effects of water activity on an enzymatic reaction, there is a need for practical methods to adjust the water activity in the reaction mixture. Likewise, it is highly desirable to keep this parameter close to the optimal value during large-scale conversions. A range of water activity control methods have been developed [11], and which one to choose depends on the scale of reaction, the quantities of water to be removed or added, and availability of equipment. 

On the other hand, the enzymatic synthesis of glycoconjugates and oligosaccharides leads to high product yields in a short time by stereo- and regioselective one-step reactions. All enzymatic reactions are easy to scale up and are carried out in aqueous media under mild conditions. A whole set of enzymes is now available to build up OAT bonds in monosaccharides, COP bonds in activated monosaccharides e.g. phosphorylated sugars or nucleotide sugars, and C-O-C bonds in di- and oligosaccharides (Fig. 1). However, all these enzymatic reactions are limited by the substrate spectrum of the individual enzyme. 

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