One-Pot Synthesis of Levulinic Acid/Ester from C5 Carbohydrates in a Methanol Medium

A process for direct conversion of xylose to methyl levulinate and levulinic acid has been developed in this study. A methanol medium, solid acid catalyst Amberlyst 70, and hydrogenation catalyst Pd/Al2O3 were used to direct xylose to follow the designed route to the target products. The methanol medium can prevent the hydrogenation of xylose to xylitol via the transformation of xylose into methyl xylosides. Amberlyst 70 catalyzes the dehydration of methyl xylosides into furfural, while Pd/Al2O3 catalyzes the hydrogenation of furfural into furfuryl alcohol. The hydrolysis/methanolysis of furfuryl alcohol over Amberlyst 70 produces levulinic acid/ester. Among these steps, the hydrogenation of furfural is the one determining the overall selectivity from xylose to levulinic acid/ester. The ideal hydrogenation catalyst needs to be selective to hydrogenate only the carbonyl group of furfural but not the furan rings

High stability and low competitive inhibition of thermophilic Thermopolyspora flexuosa GH10 xylanase in biomass-dissolving ionic liquids

Thermophilic Thermopolyspora flexuosa GH10 xylanase (TfXYN10A) was studied in the presence of biomass-dissolving hydrophilic ionic liquids (ILs) [EMIM]OAc, [EMIM]DMP and [DBNH]OAc. The temperature optimum of TfXYN10A with insoluble xylan in the pulp was at 65-70 °C, with solubilised 1 % xylan at 70-75 °C and with 3 % xylan at 75-80 °C. Therefore, the amount of soluble substrate affects the enzyme activity at high temperatures. The experiments with ILs were done with 1 % substrate. TfXYN10A can partially hydrolyse soluble xylan even in the presence of 40 % (v/v) ILs. Although ILs decrease the apparent temperature optimum, a surprising finding was that at the inactivating temperatures (80-90 °C), especially [EMIM]OAc increases the stability of TfXYN10A indicating that the binding of IL molecules strengthens the protein structure. Earlier kinetic studies showed an increased Km with ILs, indicating that ILs function as competitive inhibitors. TfXYN10A showed low increase of Km, which was 2-, 3- and 4-fold with 15 % [EMIM]OAc, [DBNH]OAc and [EMIM]DMP, respectively. One reason for the low competitive inhibition could be the high affinity to the substrate (low Km). Xylanases with low Km (~1 mg/mL) appear to show higher tolerance to ILs than xylanases with higher Km (~2 mg/mL). Capillary electrophoresis showed that TfXYN10A hydrolyses xylan to the end-products in 15-35 % ILs practically as completely as without IL, also indicating good binding of the short substrate molecules by TfXYN10A despite of major apparent IL binding sites above the catalytic residues. Substrate binding interactions in the active site appear to explain the high tolerance of TfXYN10A to ILs