High expression of zearalenone degrading enzyme in Pichia pastoris

Abstract:

High expression of zearalenone (ZEN) degrading enzyme gene (zlhy-6) in Pichia pastoris strain GS115 was achieved by codon optimization and multi-copy construction in vitro. The codon-optimized zlhy-6 gene sequence was synthesized with the alpha factor signal peptide coding sequence and inserted into the pAO815 plasmid. The expression plasmid containing 1–6 expression cassettes was constructed by enzyme digestion and transferred into P. pastoris GS115 strain to obtain the ZEN degrading enzyme recombinant strain. The molecular weight of the recombinant protein was 28.9 kDa, which was consistent with the theoretical value. After 3 days of induction fermentation, the protein concentration reached the highest level and then decreased; the expression level was the highest in the induction culture at pH 5.0 and 4.5, while the expression level at other pH was very low; the expression level was the highest when 0.8% methanol was added every day and 10% inoculation was added; the expression level of four-copy transformants was the highest, and the enzyme activity reached 10 U/mL after 3 days of flask fermentation, The degradation rate of ZEN in 1 g corn ballast was 44.08%–75.51% when 0.1–0.5 mL fermentation supernatant added and hydrolyzed for 24 hours. The results of this study laid a foundation for improving the industrial fermentation level of ZEN degrading enzyme and its application in eliminating ZEN in food and feed.