Mota Pacheco, Luis Erick; (2020) Biocatalytic conversion of renewable feedstocks for production of value-added chemicals. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Biocatalytic processes using transketolase have previously been developed in order to produce polyalcohols; an important class of chiral molecule, often used for the synthesis of active pharmaceutical ingredients. Transketolase catalyses asymmetric carbon-carbon bond formation by transferring a two-carbon moiety between a ketose and an aldose sugar. Nowadays, there is a focus on sustainable processes, based on the use of renewable feedstocks, in order to reduce energy consumption and waste production. The aim of this study was to establish novel methods for the evaluation and optimisation of the transketolase-catalysed upgrading of L-arabinose and D-galacturonic acid, the major pectin components of sugar beet pulp, a by-product obtained from sugar beet bio-refineries, and to explore their subsequent amination. The first objective involved transketolase production, activity evaluation and the development of a detailed kinetic model for the bioconversion of L-arabinose to L-glucoheptulose using cell lysates. Optimising cell growth and protein expression for the H461Y transketolase mutant and using clarified lysate as biocatalyst, an initial reaction rate of 51 µmol L-1 min-1 was reached. This is the highest initial reaction rate for the production of L-glucoheptulose reported to date. It was found that the high Michaelis constant of L-arabinose determines the rate of the overall reaction, and that Lithium hydroxypyruvate is inhibitory at concentrations > 1 mM. Screening of a library of transaminase enzymes identified a transaminase that could convert L-glucoheptulose to (2S,3S,4S,5R)-6-aminoheptane-1,2,3,4,5,7-hexaol. A kinetic model for the L-arabinose bioconversion using pure H461Y transketolase as biocatalyst was also generated. Comparison of the kinetic parameters of the purified and lysate forms of the enzyme indicated that there is no significant difference between the two enzyme preparations. A second objective was to assess the utilisation of transketolase for the upgrading of D-galacturonic acid. Mutant H461Y was identified as also being able to achieve the bioconversion of D-galacturonic acid into 2,3,4,5,6,8-hexahydroxy-7-oxooctanoic acid. A detailed kinetic model of this bioconversion was also established. This showed that H461Y transketolase presents higher affinity for D-galacturonic acid than for L-arabinose since D-galacturonic acid bioconversions reached completion in less than 12 hours. The model showed that Lithium hydroxypyruvate exhibits inhibition to the reaction at concentrations > 1 mM. This is consistent with the L-arabinose bioconversion study. Optimum conditions for 2,3,4,5,6,8-hexahydroxy-7-oxooctanoic acid synthesis would involve the use of high D-galacturonic acid and low Lithium hydroxypyruvate concentrations. Based on the insights of the kinetic model for the lysate TK, options to improve the productivity of the bioconversion process were finally explored. The most productive conditions for the synthesis of L-glucoheptulose in a fed-batch process at preparative scale involved using high L-arabinose (111 mM) and low Lithium hydroxypyruvate (>10 mM) concentrations. Under the optimised conditions the maximum L-glucoheptulose productivity achieved was 56.8 mg L-1h-1. Overall, this work has established the foundations for the biocatalytic upgrading of L-arabinose and D-galacturonic acid from SBP as a sustainable feedstock. The products formed have potential applications in hypoglycaemia and cancer treatment.

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