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Author González Torres, Jordi |
Abstract Lignocellulosic biomass is a widely available feedstock and comprises the only large-volume renewable material that is composed of aromatic compounds. Aromatic compounds have been widely studied for its properties as platform molecules, that is, molecules that can potentially be converted to high-value products. The European SMARTBOX project aims at developing a biorefinery process to revalorise platform chemicals extracted from lignocellulosic biomass. The fractionation of the biomass by Reductive Catalytic Fractionation (RCF) yields a low-molecular-weight lignin oil composed by a range of aromatic molecules, some of them considered platform chemicals. Dihydrosinapyl alcohol (DHSA) is a monolignol obtained in high yields by RCF. Within the SMARTBOX framework, this thesis portrays the efforts to develop a two-step biocatalytic transformation of DHSA to syringaresinol (SR) using an engineered eugenol oxidase (EUGO 8ES) for the first stage (DHSA to sinapyl alcohol, SA) and a commercial horseradish peroxidase (HRP) for the second stage (SA to SR). To this end, EUGO 8ES was produced in E. coli BL21(DE3) in a fed-batch fermentation and downstream processed to three different grades: resting cells (RC), cell-free extract (CFE) and purified enzyme (PE). On the other hand, the monomeric fraction of lignans was isolated by reverse phase chromatography from a DHSA-rich crude lignin oil (CLO), obtaining the purified lignin oil (PLO). Initially, syringaresinol was successfully synthesized employing the mentioned twostage one-pot strategy at low SR/DHSA yields (~40%) using CLO as substrate. The yields were further improved by adding the unspecific HRP once the first stage had been completed (late addition), e.g.: all DHSA had been converted to SA. The overall yield was almost doubled (71.7%) using PLO as substrate and EUGO 8ES CFE, reaching a final concentration of 2.7 g/L of syringaresinol. Additionally, a non-HRP-catalysed formation of SR was observed in all bioconversions with late HRP addition. This spontaneous formation of syringaresinol has not been described in literature and it is responsible for a large proportion of the SR formed in the best yielding bioconversions. |
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Director Alcaraz, Estefanía (Grifols) |
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Degree IQS SE - Master’s Degree in Bioengineering |
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Date 2022-07-09
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