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

J Agric Food Chem. 2018 Apr 25;66(16):4182-4188. doi: 10.1021/acs.jafc.8b00551. Epub 2018 Apr 12.

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.

Keywords: active aggregates; heat tolerance; purification; self-assembly amphipathic peptide; β-xylosidase.

MeSH terms

  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / isolation & purification*
  • Bacterial Proteins / metabolism
  • Enzyme Stability
  • Hot Temperature
  • Hydrogen-Ion Concentration
  • Hydrolysis
  • Kinetics
  • Peptides / chemistry*
  • Peptides / metabolism
  • Thermoanaerobacterium / chemistry
  • Thermoanaerobacterium / enzymology*
  • Thermoanaerobacterium / genetics
  • Xylosidases / chemistry*
  • Xylosidases / genetics
  • Xylosidases / isolation & purification*
  • Xylosidases / metabolism

Substances

  • Bacterial Proteins
  • Peptides
  • Xylosidases
  • exo-1,4-beta-D-xylosidase