Du, C; (2014) Bioconversion of lignin degradation products into value-added chemicals. Doctoral thesis , UCL (University College London).

Abstract

Lignin is an essential component of the cell wall of various types of plants and represents an abundant and renewable natural resource. Both thermo-chemical and biological pre-treatment can be applied to break down the strong ether bonds and phenylpropanoid polymer subunits present in lignin. These liberate a range of phenolic compounds which represent potential substrates for bioconversion by ω-transaminases (ω-TAm). In this work the utility of the CV2025 ω-TAm from Chromobacterium violaceum DSM30191 is explored for selective amination of lignin breakdown intermediates into value-added products. Eight potential ω-TAm substrates were initially screened using (S)-α-methylbenzylamine (MBA) as the amino donor. Vanillin was identified as the best potential substrate which is converted into vanillylamine, an intermediate in the preparation of pelargonic acid vanillylamide used as a hyperemia inducing active substance in wound dressings. At low vanillin and MBA concentrations (<10mM) and with an excess of the amine donor (1:4 mol/mol) 100% w/w conversion of vanillin into vanillylamine was observed within 60 min. At vanillin concentrations above 10 mM, inhibition was observed, decreasing the rate and yield of the bioconversion. A kinetic model of the bioconversion was subsequently established, based on the ping-pong bi-bi mechanism, which indicated that the reaction product, vanillylamine and by-product (acetophenone) were also inhibitory. Mutant libraries prepared by saturation mutagenesis at ten amino acid residues located in the active site of the CV2025 TAm enzyme were next evaluated in order to enhance the activity and increase the substrate range. A microwell-based high-throughput screening approach (300 µL scale) identified one particular mutant, R416Q, that increased both the rate of the vanillin bioconversion and those involving substrates (acetovanillone and p-hydroxyacetophenone) that would lead to formation of chiral amine products. For the vanillin bioconversion, the R416Q mutant displayed reduced inhibition from both vanillylamine product and the acetophenone by-product. Finally, fed-batch operation in larger scale, pH-controlled, stirred bioreactors (15 mL) was examined in order to overcome vanillin substrate inhibition. With the wild-type ω-TAm fed-batch bioconversions (feeding high concentrations of both vanillin and MBA) enabled a doubling of the final product concentration obtained. Introduction of the R416Q mutant ω-TAm with the fed-batch process enabled a further 3-fold increase in the final product concentration. Through this combination of enzyme engineering and bioprocess engineering technologies a 93.8% w/w substrate conversion was achieved at a final product concentration of 17.3 g.L-1 (112.6 mM) after three consecutive batches within 12 hr. These results demonstrate the potential for bioconversion of lignin breakdown products into value-added chemicals but illustrate the need for enzymes with improved substrate range and reduced sensitivity to product inhibition.

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