Increasing pressure mandated by different government policies, for developing sustainable chemical processes for the synthesis of optically pure compounds, has resulted in increased considerations of biocatalysis as a viable option by many industries. Biocatalysis, with its exquisite selective properties and potential ‘green’ attributes, presents it as a sustainable alternative. Today, the role of biocatalysis is most evident in the pharmaceutical industry and is currently extending towards fine and bulk chemical production as well. The use of hydrolytic enzymes (lipases) is well established in several chemical industries, though certain challenges persist in other types of enzymes (transferases and ketoreductases), thus limiting their implementation in industry. Inhibition by substrate and product as well as low aqueous solubility of substrates has constrained the full potential of these enzymes to be harnessed. Porous resins as opposed to other auxiliary phases, for example organic solvents, are nonbioavailable, biocompatible and offer simpler operational handling (no foaming and emulsification). This strategy has been applied effectively to single substrate – single product systems (oxidation, V microbial degradation and hydrolysis). However, this concept has not been extended to other industrially relevant reactions which are two substrate – two product systems. In this thesis, a methodological framework has been successfully developed to aid in implementing the strategy of integrating porous resins for multi-component systems. In this manner, a generic platform has been established for biocatalytic reactions that require the integration of this strategy. The framework identifies the key information about the reaction and the process using a step-wise protocol with the required tools. It includes the use of kinetic modelling in characterizing the reaction kinetics, a heuristic approach for screening resins and a model based approach for evaluating the process. Greater knowledge about the enzymatic processes with integrated porous resins can therefore be gained and thus the efficiency of process development with respect to time and resources required (reduced number of experiments) could be increased. Estimating kinetic model parameters for enzymatic reactions is quite complex and frequently leads to identifiability issues. In order to understand the different techniques to estimate the parameters, a number of concepts are discussed in chapter four of this thesis. This knowledge has contributed to the development of a robust methodology for the estimation of kinetic model parameters for biocatalytic reactions, which has also been published in a peer reviewed journal. Screening resins for moderately hydrophobic multi-component systems is challenging. Often it is found that the capacity of the resin is inversely related with product selectivity. Therefore a tradeoff has to be made between these parameters which can be crucial from an economic point of view. A low resin capacity points towards the need for higher resin loading, which in turn determines the equilibrium concentration of the substrate in the reactor and the type of reactor that can be used (stirred tank reactor or packed bed reactor). Similarly low product selectivity would result in higher product concentration in the reactor and thus not aid in alleviating inhibition. Further considerationsProcess modelling is a very effective tool in evaluating a process. Critical information about the process can be gained by means of simulations, which can further be re-used to tune the reaction or process conditions to harness the full potential of the enzyme. State-of-the-art mathematical techniques for model quality evaluation, such as uncertainty and sensitivity analysis, have been included in this analysis in order to identify the key model parameters for better understanding of the process. Three case studies were used to illustrate the applicability of the methodology to fulfil different objective requirements. The case studies were selected for not only being industrially relevant but as well as having certain limitations which contributed in developing the tools and strategies to overcome them. The asymmetric synthesis of 1-phenylethylamine using Ȧ-transaminse, the asymmetric synthesis of 1-methyl-3-phenylpropylamine using Ȧ-transaminse and enantioselective synthesis of 2-octanol using alcohol dehydrogenase were selected. VI of resin stability and cost also have to be taken into account in the screening procedure. The screening therefore becomes a multi-objective task that has to be solved simultaneously. Such an approach has been applied in the method formulated in this framework. To overcome these challenges, different process strategies are required to obtain high yields. A number of different challenges and proposed solutions are discussed in chapter one of this thesis and have also been published as a review. In recent years, integrating porous resins as an auxiliary phase in enzymatic processes, to non-selectively bind the substrate and product as a means to alleviate substrate and product inhibition, has gained considerable recognition. The resins act as a reservoir for the inhibitory substrate and a sink for the inhibitory product and simultaneously attain the required high substrate loading to make the process economically feasible. In this way the potential benefit of the enzyme can be exploited.
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Tufvesson, Pär, Gani, Rafiqul, Woodley, John
Technical University of Denmark, Department of Chemical Engineering, 2012