β-Cyclodextrin and ultrasound-assisted enzyme renaturation for foam fractionation of laccase from fermentation broth of Trametes hirsuta 18

Abstract

Foam fractionation is a cost-effective method to separate laccase from its fermentation broth. However, the enzyme readily suffered a substantial activity loss in the separation process. To reduce the activity loss, we firstly analyzed the structure-function relationship of laccase to explain how laccase was denatured in the foam fractionation of laccase from fermentation broth of Trametes hirsuta 18 using cetyltrimethyl ammonium bromide (CTAB) as a collector. The results demonstrate that via electrostatic force, CTAB interacted with laccase molecules to cause their structural unfolding, so CTAB resulted in the denaturation of laccase. Increasing the molar ratio of CTAB vs laccase at the gas-liquid interface over that in the feed solution, foaming enhanced the CTAB-induced denaturation. On this ground, we attempted to use β-cyclodextrin and ultrasound to assist the refolding of laccase denatured jointly by CTAB and foaming in the foamate. Ultrasound effectively assisted the renaturation of denatured laccase after it was separated from CTAB by β-cyclodextrin in 40 min at 20 °C or lower. Furthermore, their renaturation performances highly depended on pH, volumetric airflow rate, CTAB concentration and molar ratio of β-cyclodextrin vs CTAB in the foamate. At pH 6.0, volumetric airflow rate 60 mL/min, CTAB concentration 0.4 g/L and molar ratio of β-cyclodextrin vs CTAB in the foamate of 1.6:1, the enrichment ratio and recovery yield of laccase activity and purification fold of laccase reached 73.4%, 11.9 and 3.5, respectively. They were 230% higher than those without β-cyclodextrin and ultrasound. Therefore, ultrasound and β-cyclodextrin effectively enhanced the CTAB-assisted foam fractionation of laccase from the fermentation broth of T. hirsuta 18. In addition, a SDS-PAGE analysis determined that laccase of 55 kDa in molecular weight was highly enriched by the foam fractionation.