Design of a highly thermostable hemicellulose-degrading blend from Thermotoga neapolitana for the treatment of lignocellulosic biomass

The biological conversion of lignocellulose into fermentable sugars is a key process for the sustainable production of biofuels from plant biomass. Polysaccharides in plant feedstock can be valorized using thermostable mixtures of enzymes that degrade the cell walls, thus avoiding harmful and expensive pre-treatments. (Hyper)thermophilic bacteria of the phylum Thermotogae provide a rich source of enzymes for such industrial applications. Here we selected T. neapolitana as a source of hyperthermophilic hemicellulases for the degradation of lignocellulosic biomass. Two genes encoding putative hemicellulases were cloned from T. neapolitana genomic DNA and expressed in Escherichia coli. Further characterization revealed that the genes encoded an endo-1,4-β-galactanase and an α-L-arabinofuranosidase with optimal temperatures of ˜90 °C and high turnover numbers during catalysis (k cat values of ˜177 and ˜133 s −1 , respectively, on soluble substrates). These enzymes were combined with three additional T. neapolitana hyperthermophilic hemicellulases – endo-1,4-β-xylanase (XynA), endo-1,4-β-mannanase (ManB/Man5A) and β-glucosidase (GghA) – to form a highly thermostable hemicellulolytic blend. The treatment of barley straw and corn bran with this enzymatic cocktail resulted in the solubilization of multiple hemicelluloses and boosted the yield of fermentable sugars by up to 65% when the complex substrates were further degraded by cellulases.