Functional characterization of a novel thermophilic exo-arabinanase from Thermothielavioides terrestris.

Arabinanases from glycoside hydrolase family GH93 are enzymes with exo-activity that hydrolyze the α-1,5 bonds between arabinose residues present on arabinan. Currently, several initiatives aiming to use byproducts rich in arabinan such as pectin and sugar beet pulp as raw material to produce various compounds of interest are being developed. However, it is necessary to use robust enzymes that have an optimal performance under pH and temperature conditions used in the industrial processes. In this work, the first GH93 from the thermophilic fungus Thermothielavioides terrestris (Abn93T) was heterologously expressed in Aspergillus nidulans, purified and biochemically characterized. The enzyme is a thermophilic glycoprotein (optimum activity at 70 °C) with prolonged stability in acid pHs (4.0 to 6.5). The presence of glycosylation affected slightly the hydrolytic capacity of the enzyme, which was further increased by 34% in the presence of 1 mM CoCl2. Small-angle X-ray scattering results show that Abn93T is a globular-like-shaped protein with a slight bulge at one end. The hydrolytic mechanism of the enzyme was elucidated using capillary zone electrophoresis and molecular docking calculations. Abn93T has an ability to produce (in synergism with arabinofuranosidases) arabinose and arabinobiose from sugar beet arabinan, which can be explored as fermentable sugars and prebiotics. KEY POINTS: • Thermophilic exo-arabinanase from family GH93 • Molecular basis of arabinan depolymerization.

Heterologous expression and functional characterization of a GH10 endoxylanase from Aspergillus fumigatus var. niveus with potential biotechnological application.

Xylanases decrease the xylan content in pretreated biomass releasing it from hemicellulose, thus improving the accessibility of cellulose for cellulases. In this work, an endo-ß-1,4-xylanase from Aspergillus fumigatus var. niveus (AFUMN-GH10) was successfully expressed. The structural analysis and biochemical characterization showed this AFUMN-GH10 does not contain a carbohydrate-binding module. The enzyme retained its activity in a pH range from 4.5 to 7.0, with an optimal temperature at 60 °C. AFUMN-GH10 showed the highest activity in beechwood xylan. The mode of action of AFUMN-GH10 was investigated by hydrolysis of APTS-labeled xylohexaose, which resulted in xylotriose and xylobiose as the main products. AFUMN-GH10 released 27% of residual xylan from hydrothermally-pretreated corn stover and 14% of residual xylan from hydrothermally-pretreated sugarcane bagasse. The results showed that environmentally friendly pretreatment followed by enzymatic hydrolysis with AFUMN-GH10 in low concentration is a suitable method to remove part of residual and recalcitrant hemicellulose from biomass.