Biocatalyst engineering applied to the synthesis of fatty xylose esters derived from the C-5 fraction of lignocellulosic residues

Abstract

The growing demand for sustainable, low-environmental-impact surfactants has driven the development of biotechnological routes capable of converting renewable feedstocks into high-value products. In this context, the enzymatic synthesis of xylose fatty acid esters stands out as a promising alternative, particularly due to its potential do valorize the C5 fraction of lignocellulosic biomass, which remains underexploited in industrial applications. However, challenges related to low solubility of xylose, the limited stability of biocatalysts, and reduced catalytic efficiency in non-conventional media still hinder the implementation of these processes. This thesis aimed to develop and evaluate integrated strategies for the enzymatic production of xylose esters, addressing key gaps in biocatalyst engineering, reaction medium selection, and the use of biomass-derived feedstocks. Initially, the state of the art in the enzymatic synthesis of xylose esters was assessed through systematic literature mapping, enabling the identification of recurring strategies, limitations, and opportunities related to catalysts, acyl group donors, solvents, and downstream processing. The results indicated that lipases are the preferred biocatalysts for xylose ester synthesis. Additionally, solvent selection was identified as a crucial factor, with oleic acid, lauric acid, and methyl and vinyl esters being the most commonly employed acyl donors. Subsequently, post-treatment strategies were investigated for the commercial biocatalyst Novozym® 435. The results demonstrated that coating with polyethyleneimine followed by crosslinking with glutaraldehyde yielded more robust biocatalysts, resulting in a 2.3-fold increase in oleic acid conversion when a deep eutectic solvent was used to solubilize xylose, and a 1.5-fold increase when methyl ethyl ketone and tert-butanol were employed. In addition, post-treatment improved operational stability over successive reaction cycles, with approximately 75% retention of catalytic activity after five reuse cycles. A key advancement of this work was the use of a chloride–xylose deep eutectic solvent, which significantly enhanced xylose solubility, reaching concentrations above 600 mM, substantially higher than those observed in conventional organic solvents (7 mM in methyl ethyl ketone and 60 mM in tert-butanol). This increased solubility directly improved enzymatic performance, enabling higher conversion rates and high selectivity toward xylose monoesters. In parallel, the effects of different immobilization protocols and metal modification of the lipase Eversa® Transform 2.0 were evaluated, revealing variations in catalytic activity, thermal stability, and specificity. Spectroscopic analyses confirmed conformational changes in the immobilized enzymes, allowing correlation between immobilization pH and the observed hydrolytic activity and catalytic specificity. Finally, the feasibility of producing xylose esters from sugarcane bagasse was demonstrated through the integration of biomass pretreatment and enzymatic conversion steps. Pretreatment with acetic acid and hydrogen peroxide resulted in a fourfold increase in sugar release during enzymatic hydrolysis with xylanases, compared to untreated bagasse. Biocatalysts prepared by immobilizing Eversa® Transform 2.0 at pH 5 in the presence of 100 mM NaCl, followed by metal modification with MgCl₂, were evaluated. The non-metal-modified biocatalyst exhibited superior performance, achieving 15% oleic acid conversion and 63% xylose conversion. Overall, the modulation of biocatalyst properties through immobilization and post-treatment strategies, the use of deep eutectic solvents in xylose esterification, and the application of xylose derived from sugarcane bagasse significantly advance the field of applied biocatalysis for sugar ester synthesis. This findings provide concrete perspectives for the development of sustainable industrial routes for biosurfactant production.