Multienzyme process development

Perhaps the first decision to be made in process development is the difficult decision of whether the enzymes to be used should be used in an integrated format. Such a question does not arise with conventional single biocatalytic steps but is highly important in multienzyme processes. One of the key criteria here is whether the enzymes can be operated together without compromise to any of the individual enzyme s activity or stability. An interaction matrix (see Section 10.6) can be used to assist such decision making. In cases where the cost of one or more of the enzyme(s) is not critical, it will be possible to combine in a one-pot operation. In other cases, where the cost of an individual enzyme becomes critical, then it may be necessary to separate the catalysts, such that each can operate under optimal conditions. Likewise, selection of the biocatalyst format (immobilized enzyme, whole cell, cell-free extract, soluble enzyme, or combinations thereof) in combination with the basic reactor type (packed bed, stirred tank, or combinations thereof) and biocatalyst recovery (mesh, microfiltration, ultrafiltration, or combinations thereof) will determine the structure of the process flowsheet and therefore is an early consideration in the development of any bioprocess. The criterion for selection of the final type of biocatalyst and reactor combination is primarily economic and may best be evaluated by the four metrics in common use to assess the economic feasibility of biocatalytic processes [29] ... 

For a multienzyme process, this evaluation is critically important to achieve a better theoretical understanding of the process and to achieve useful modeling and process design. The reaction considerations describe the key characteristics to understand how the interaction between enzymes and components can be interpreted for modehng. Furthermore, such information forms the basis for the formulation of reaction rates for the different enzymes that are involved in multienzyme process. In this manner, a preliminary idea of enzyme mechanisms and kinetic parameters, that can be expected when developing a model, may be obtained. Key information (Figure 10.3) may be described as... 

We finally believe that novel methodologies of multienzyme processes for dynamic resolutions will be developed, whereas the applications of DKR in organic synthesis will continue to fascinate researchers in academia as well as in pharmaceutical industry. 

Cascade architecture A graphical representation of all reactions in the mulhen-zyme process is the basis for describing the final model structure. It includes the primary reachons, secondary reactions, and competing reactions. For a single enzyme, reachon mechanisms are well developed, and they are then included into the full model to describe the multienzyme process by combining the effect of the individual enzymes. In this way, the different possible reaction schemes are generated to give the cascade structure. 

Nature, however, has performed more than simple stepwise transformations using a combination of enzymes in so-called multienzyme complexes, it performs multistep synthetic processes. A well-known example in this context is the biosynthesis of fatty acids. Thus, Nature can be quoted as the inventor of domino reactions. Usually, as has been described earlier in this book, domino processes are initiated by the application of an organic or inorganic reagent, or by thermal or photochemical treatment. The use of enzymes in a flask for initiating a domino reaction is a rather new development. One of the first examples for this type of reaction dates back to 1981 [3], although it should be noted that in 1976 a bio-triggered domino reaction was observed as an undesired side reaction by serendipity [4]. 

Multienzyme modular assemblies such as PKSs and NRPSs have flexible swinging tethers which channel covalently bound intermediates between successive active sites. Swinging arms plus specific protein-protein interactions offer mechanisms for the transfer of substrates between modules and offer concepts for the development of one-pot multi-reaction biocatalytic processes. 

Since enzymes generally function under the same or similar conditions, several biocatalytic reactions can be carried out in a reaction cascade in a single flask. Thus, sequential reactions are feasible by using multienzyme systems in order to simplify reaction processes, in particular if the isolation of an unstable intermediate can be omitted. Furthermore, an unfavorable equilibrium can be shifted towards the desired product by linking consecutive enzymatic steps. This unique potential of enzymes is increasingly being recognized as documented by the development of multienzyme systems, also denoted as artificial metabolism [15]. 

Fatty acid biosynthesis utilizes acetyl CoA. Radioactive acetate is the common experimental substitute, but in the developing seed sucrose from the mother plant is the initial source of substrate. Biosynthesis is a multi-step process (Fig. 3.14) which firstly involves the formation of malonyl CoA by carboxylation of acetyl CoA with carbon dioxide. This malonyl CoA is then accepted by acyl carrier protein (ACP) which is part of a multienzyme complex called the ACP fatty acid synthetase complex. The malonyl CoA is then condensed with... 

Self-sorting is one of the most fundamental processes in living systems, which led complex mixtures of biomolecules (e.g., proteins, nucleic acids, oligosaccharides, lipids) to self-organize into larger biomacromolecular assemblies (e.g., DNA double-helix, multienzyme complexes, ribosomes) and finally into cellular compartments essential for the development of life on Earth. 

In all of the multistep immobilized enzyme work done to date, theoretical or experimental, for modelling purposes or for applications, there exists one common factor the chemical reactions are affected by the diffusive processes so that the macroscopically observed kinetics are strongly perturbed by the incorporation of the enzymes into a gel. This perturbation is caused by the development of localized concentrations and concentration gradients within the gel which are quite different from that found in free solution. Only one instance appears to have been reported where exact modelling of real experimental data has been attempted. All other work has been either purely theoretical or qualitative interpretations of limited experimental data. There is still much to be learned of the role played by the gel matrix in affecting the overall kinetic performance of gel entrapped multienzyme systems before they can be well designed for applications or used with any confidence in a quantitative way as models for living systems. 

---