A novel method for hydrophobin extraction using CO2 foam fractionation system

Due to the exceptional properties and many potential applications of hydrophobins, special fungal proteins, it becomes necessary to develop a real scale procedure for their production and purification. In our previous study (Deckers et al., 2010) [CO ₂-hydrophobin structures acting as nanobombs in beer, Brew. Sci. 63:54-61], the strong interaction of CO ₂-hydrophobin was demonstrated. For the first time, in an approach to isolate hydrophobin HFBII from the growth media of Trichoderma reesei, a foam fractionation system using CO ₂ as the sparging gas was investigated in this study. Using CO ₂ foam fractionation, the concentration of HFBII was increased from 0.10±0.02mg/mL up to 0.57±0.04mg/mL. This was shown after a purification step by conventional liquid chromatography and identification of the goal protein using MALDI-TOF. The obtained molecular weight of the protein was 7.042kDa which corresponds to the complete molecule of HFBII, minus the last aminoacid. Micro-spectrophotometry was used for quantification of purified HFBII. Moreover, different parameters of the foam fractionation system were optimized. The concentration of the protein after treatment by CO ₂ followed by liquid chromatography was increased from 0.32±0.02 to 0.44±0.06mg/mL when the flow rate of gas injection was changed in the range of 1-3L/min. The highest amount of HFBII equal to 0.57±0.04mg/mL was obtained by the highest ratio of liquid height over the column height. Using the larger pore size frits causes increased protein absorption as well. The gushing potential of samples revealed that in contrast to the samples before CO ₂ treatment, interestingly, no gushing was observed for the samples after treatment. The possibility that stable aggregates of HFBII molecules are formed as a consequence of their high concentration is discussed in this paper. By using DLS analysis of the overfoam, 100nm particle size of CO ₂ nanobubbles coated by HFBII was obtained. The final concentration of the protein was carried out using Amicon ^® ultracentrifuge device with the average recovery of 63.8±8.2%.