| Summary: | Recently interest in biocatalysis, the use of enzymes as catalysts, has been growing. This is due in part to the ability to achieve new and interesting chemistry, but also more sustainable, milder, aqueous processes producing less waste. With these advantages though, come new process bottlenecks. Many methods of overcoming such disadvantages have been proposed. However, key to the designer is the ability to discriminate between them at an early stage. To date several methods of early identification of bioprocess bottlenecks have been presented, though none of these has enabled the easy identification of the biocatalyst requirement in the reactor with regard to integrating upstream and downstream requirements. To this end a tool by which biocatalytic processes can be analysed and evaluated, based in part on regime analysis has been presented. The method identifies biocatalyst concentrations at which limitations that control chosen process metrics become dominating. The tool has been demonstrated by the use of a model system, the biocatalytic production of S,S- ethylenediamine-N.N'-disuccinic acid by two enzymes, EDMSase and EDDSase. The reaction is characterised by fumarate inhibition, an exotherm, competition for fumarate not least by the two half reactions, equilibria close to unity and currently an unproductive wild type fermentation. Key bottlenecks were determined to be a severe limitation by the enzyme catalysing the first step, a fumarase limitation and an equilibrium limitation. Proposal of metric hurdles enabled analysis of the dominating regimes and led to determination of the feasible operating region at 232 g.l"1 fumarate, 30 g.P1 ethylenediamine, 900 g < fcw.r1 EDDSase and 300 g.I"1 EDMSase, enzyme concentrations unachievable by current methods. Possible processes and research to overcome these limitations were proposed and analysed for their effect on the metrics indicating that in the absence of fumarase, use of in-situ product removal would improve process yield 2-fold and product concentration 3-fold.
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