This work is a response to the crescent interest by the biorefinery industries that drive research towards more cost-competitive and environmentally sustainable processes. New ways to value materials that would once be considered wasteful or valueless are researched. Lignin is one of such cases, a complex biopolymer that is considered by the paper and ethanol producing industries as waste. Most importantly, lignin is the only large-volume renewable feedstock that contains aromatic compounds, making it an attractive candidate for research efforts towards its valorization. In this study, isoeugenol, a compound resultant from lignin reductive catalytic fractionation (RCF) was used as substrate for vanillin production. This bioconversion was achieved resorting to an isoeugenol monooxygenase as biocatalyst. At start, the heterologous production of two isoeugenol monooxygenases, one from Pseudomonas putida (PpIEMO) and one from P. nitroreducens (PnIEMO) were compared regarding protein solubility and enzyme activity, in 50 mL scale cultures using different E. coli strains. The isoeugenol monooxygenase from P. nitroreducens was then picked to proceed to scale-up and characterization processes using E. coli Tuner ΔcueO as host. After production at 1L-scale, PnIEMO was purified by affinity chromatography techniques and biochemically characterized to assess its potential application in biotechnology. The evaluating of its properties and limitations will allow in the future, to proceed to enzyme evolution. Purification of PnIEMO from 1 L of growth medium yielded ≈ 1.8 mg of purified enzyme. The molecular mass determined was of 50.1 kDa. The optimum pH and temperature for this enzyme were pH 9 and 30 °C. Michaelis Menten parameters were calculated and resulted in Km and Km/Kcat of 0.13 mM and 118 mM-1 s-1, respectively. PnIEMO presented low stability towards temperature, with half-life of 7 min at 35 °C and a melting temperature of 32 °C (temperature at which 50 % of the molecules are unfolded), when following tryptophan fluorescence as protein denaturation indicator. This enzyme presented some tolerance towards guanidine hydrochloride, displaying a midpoint of the concentration of 1.3 M (where 50 % of molecules are unfolded). The techniques assessed to improve the yields of soluble enzyme, namely the use of hosts co-expressing chaperones, or to recover active enzyme from the inclusion bodies present in the cell pellets, by unfolding and refolding processes, did not show improvements. Whole-cell assays were performed using free and alginate immobilized E. coli cells expressing PnIEMO towards isoeugenol bioconversion to vanillin. Both approaches enabled ≈ 100 % vanillin production in 24 h, although using free whole-cells the reaction was faster than with alginate immobilized whole-cells. Summing up, this enzyme is an excellent candidate for use in biotechnological vanillin production processes, using isoeugenol resultant from lignin RCF.

Tese de mestrado, Microbiologia Aplicada, Universidade de Lisboa, Faculdade de Ciências, 2019