Urea amidolyase displayed on the surface of Saccharomyces cerevisiae for ethyl carbamate reduction in Chinese Huangjiu

Abstract

Chinese Huangjiu, as one of the most popular traditional alcoholic beverages, poses a series of potential food safety issues due to its relatively high content of the “2A” class carcinogen ethyl carbamate (EC). Urea in Huangjiu is the primary precursor of EC formation. To decrease the EC content in Huangjiu, the endogenous urea amidolyase (UA) of Saccharomyces cerevisiae, which can hydrolyze urea, was anchored on the cell wall by utilizing yeast surface display technology and the cells were applied to Huangjiu fermentation or added to commercial Huangjiu in this study. Four different anchoring proteins, namely Cwp2p, Sag1p, Flo1p, and Aga1p-Aga2p, were used to construct four corresponding surface display strains. Among them, ACCD strains with CAR1 knocked out and displaying UA in the cell wall by the anchoring protein Cwp2p based on AY14α, showed the best Urea and EC degradation efficiency during simulated Huangjiu fermentation. Huangjiu by achieving reductions of 89.9% and 56.4% in urea and EC contents, respectively, compared to the control strains. Furthermore, when the ACCD strain was used as an immobilized enzyme to treat commercial Huangjiu, reductions of 29.8% and 38.8% were observed in urea and EC contents, respectively. Moreover, after 10 cycles of reuse, the degradation of urea could still reach 74.53% of the first treatment. This study presents a novel strategy for the reduction of urea and EC content in Huangjiu, and provides implications for the reduction of EC and urea content in other alcoholic beverages.