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

Xu, Tianwang, Huang, Xiongliang, Li, Zhe, Ki Lin, Carol Sze, Li, Shuang
Journal of agricultural and food chemistry 2018 v.66 no.16 pp. 4182-4188
Thermoanaerobacterium aotearoense, catalytic activity, centrifugation, chromatography, enzyme activity, half life, heat inactivation, heat tolerance, hydrolysis, peptides, proteinases, reducing sugars, sugarcane bagasse, temperature, thermal stability, thermostable enzymes, washing, xylan 1,4-beta-xylosidase
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.