Rong Huang and Yuyang Song*
*College of Enology, Northwest A&F University, College of Enology, Northwest A&F University, 712100, China (yuyangsong@nwsuaf.edu.cn)

Protein turbidity is a significant defect showing non-biological instability of wine. Acid protease is often used during food fermentation because its hydrolysis process helps avoid bacterial growth and corruption. Expression of acid protease pepA was initiated using the promoter and anchor protein in the surface display system of Saccharomyces cerevisiae. The acid protease gene (pepA) from Aspergillus usamii was cloned to construct an integrated plasmid and an integrated vector to obtain the recombinant plasmid PUC-GAP-α-factor-pepA-SED1. After the recombinant plasmid is cut with Sma I, it is transformed into the haploid and diploid of S. cerevisiae using an electric shock transformation method. Through homologous recombination, the acid protease gene (pepA) of A. usamii was integrated into the gene locus of S. cerevisiae. Two strains of S. cerevisiae that secrete acid protease through a cell surface display were obtained. Their protease activities were 285.71 U/mL and 495.24 U/mL. The research outlines a new method to solve the problem of protein turbidity in wine and lays a theoretical foundation for industrial application of pepA acid protease whole- cell catalyst.

Funding Support: (1) National Key R&D Program of China (Item no. 2019YFD1002500); (2) National Natural Science Foundation of China (31501463); (3) China Agriculture Research System (grant no. CARS-29-jg-03); (4) Viticulture Experiment Station scientific and technological transformative project of Northwest A&F University (TGZX2019-27); (5) The Fundamental Research Funds for the Central Universities (2452020177).