Abstract
ABSTRACT Aqueous two-phase separation (ATPS) using nonionic surfactants is important for protein purification. Multiple nonionic surfactants with different cloud points and molecular structures are tested for pre-purification of the Class II hydrophobin HFBII, an amphiphilic protein valued for its potential in food, cosmetics and pharmaceutical industry. The effect of time, temperature and salt addition on phase separation is investigated as well as the influence of surfactant concentration on HFBII partitioning, purity and recovery. Incubation time had little effect on phase separation while, in general, an increased temperature facilitated phase separation. Addition of NaCl also facilitated phase separation but reduced HFBII purity in the surfactant-rich phase. An increased surfactant concentration improved HFBII recovery although negatively affecting HFBII purity in the surfactant-rich phase. When applying the secondary ethoxylated surfactant Tergitol 15-S-5 (5 wt%) at 40°C without NaCl, HFBII purity was enhanced 3.9 ± 0.2 times after back extraction with isobutanol, giving a HFBII recovery of 71.8 ± 2.1%. Using these most optimal conditions, the process could successfully be scaled up resulting in a further increase of HFBII recovery and enrichment to 81.1 ± 2.1% and 6.4 ± 0.3, respectively, confirming the applicability of ATPS for pre-purification of HFBII.
Published Version
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