Enhanced Purification Efficiency and Thermal Tolerance of <i>Thermoanaerobacterium aotearoense</i> β-Xylosidase through Aggregation Triggered by Short Peptides

Abstract

To simplify purification and improve heat tolerance of a thermostable β-xylosidase (ThXylC), a short ELK16 peptide was attached to its C-terminus, which is designated as ThXylC–ELK. Wild-type ThXylC was normally expressed in soluble form. However, ThXylC–ELK assembled into aggregates with 98.6% of total β-xylosidase activity. After simple centrifugation and buffer washing, the ThXylC–ELK particles were collected with 92.57% activity recovery and 95% purity, respectively. Meanwhile, the wild-type ThXylC recovery yield was less than 55% after heat inactivation, affinity and desalting chromatography followed by HRV 3C protease cleavage purification. Catalytic efficiency (Kcat/Km) was increased from 21.31 mM–1 s–1 for ThXylC to 32.19 mM–1 s–1 for ThXylC–ELK accompanied by a small increase in Km value. Heat tolerance of ThXylC–ELK at high temperatures was also increased. The ELK16 peptide attachment resulted in 6.2-fold increase of half-life at 65 °C. Released reducing sugars were raised 1.3-fold during sugar cane bagasse hydrolysis when ThXylC–ELK was supplemented into the combination of XynAΔSLH and Cellic CTec2.