Blayer, Simone; (1997) A rational approach to biotransformation process design: Chemo-enzymatic synthesis of N-acetyl-D-neuraminic acid. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

This thesis describes a rational approach to process design for biotransformation processes. The approach enabled process flow-sheet decisions to be made based on structured scientific evidence and provided key data affecting design choices. The two step synthesis of N-acetyl-D-neuraminic acid (Neu5Ac) was taken as a model for process design. Neu5Ac was synthesised starting from N-acetyl-D-glucosamine (GlcNAc) and pyruvate (Pyr) in two steps. The first was the base-catalysed epimerisation of GlcNAc to N-acetyl-D-mannosamine (ManNAc). The second step was a biotransformation, carried out by the addition of pyruvate to ManNAc to yield Neu5Ac in equilibrium. The latter reaction was catalysed by the E. coli Neu5Ac aldolase. The first phase to the structured approach to biotransformation process design was based on characterisation. A first group of experiments investigated the components and reaction properties and this set of data indicated advantageous conditions on which to focus subsequent characterisation. The second set of data concerned the interactions between reactants, product and biocatalyst at the conditions selected above. Reaction kinetics at high substrate concentrations has determined that pyruvate was strongly inhibitory on initial rates of reaction and ManNAc was found inhibitory above 750 mM. The experimental data were then used to evaluate and select different process options, based on design rationale and engineering heuristics. As a result of this approach, process operating windows have been generated, defining possible operating regions for subsequent scale-up. Conventional reactor designs and novel integrated layouts such as in situ product removal were then experimentally assessed. In addition, the option of integration of the epimerisation with the biotransformation (at alkaline pH) has been evaluated. The reactor options were then compared for product, enzyme and downstream separation limiting process scenarios. The methodology of this rational approach to process design was then discussed for the whole class of aqueous phase equilibrium controlled biotransformations. This design approach has advanced in two ways as the result of the findings in this thesis. The characterisation step has been divided further in two sets of experiments, so that unfeasible options were quickly ruled out, accelerating the design procedure. In addition, economic considerations in the form of different processing scenarios have been introduced in the identification of design constraints.

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