Improved extraction and purification of the hydrophobin HFBI

Biotechnol J. 2021 Nov;16(11):e2100245. doi: 10.1002/biot.202100245. Epub 2021 Sep 2.


Hydrophobins (HFBs) are a group of highly functional, low molecular weight proteins with the ability to self-assemble at hydrophobic-hydrophilic interfaces. The surface active, cysteine-rich proteins are found in filamentous fungi such as Trichoderma reesei. In the present study multiple extraction solvents and conditions were screened for the mycelium bound hydrophobin HFBI and the effects on the total amount of extracted proteins, HFBI recovery and HFBI gushing activity were investigated to gain a more thorough scientific insight on the extraction efficiency and selectivity. Results indicated the enhanced selectivity for HFBI extraction from the fungal biomass using 60% ethanol compared to solutions containing 1% sodium dodecyl sulphate (SDS). Complementing the higher selectivity, HFBI recovery was increased from 6.9 ± 0.6 mg HFBI (1% SDS) to 9.4 ± 0.4 mg HFBI per gram dry fungal biomass for extracts containing 60% ethanol. Furthermore, subsequent to HPLC purification, Cold Induced Phase Separation (CIPS) of acetonitrile-water systems was investigated at different pH levels. CIPS at pH 2.0 was found to effectively remove the majority of sorbicillinoid pigments from the purified HFBI fraction. The improved method resulted in a recovery of 85.4% of the extracted HFBI after final purification.

Keywords: HFBI; Trichoderma reesei; class II hydrophobin; cold induced phase separation; extraction; fast protein liquid chromatography.

MeSH terms

  • Fungal Proteins*
  • Hydrophobic and Hydrophilic Interactions
  • Hypocreales
  • Imidazoles
  • Trichoderma*


  • Fungal Proteins
  • Imidazoles
  • 1-(heptafluorobutyryl)imidazole

Supplementary concepts

  • Trichoderma reesei