Systematic optimization for high-level secretory expression of a thermostable zearalenone hydrolase ZENM in Pichia...

Zearalenone (ZEN), a mycotoxin commonly found in cereal grains, poses a significant threat to livestock health. ZEN hydrolase is considered a key tool for detoxifying ZEN contamination due to its high efficiency, cost-effectiveness, and ability to completely degrade the toxin. Among these enzymes, ZEN hydrolase ZENM is a promising candidate for industrial applications owing to its superior thermostability. In this study, ZENM was heterologously expressed in the Pichia pastoris system. The enzyme exhibited optimal activity at pH 8.0 and 60 °C, and maintained over 90 % of its relative activity within the 50-60 °C range. Its kinetic parameters, the apparent Michaelis-Menten constant (K_m, _app) and maximal reaction velocity (V_max), were determined to be 21.59 ± 7.34 μM and 0.031 ± 0.002 μM s^-1 mg^-1, respectively. The yield of ZENM was increased from 6.70 U/mL to 41.55 U/mL by utilizing the pre-Ost1-pro-α-factor signal peptide. Increasing the gene dosage further enhanced the yield to 50.87 U/mL. Furthermore, co-expression with molecular chaperones PDI and SEC12 elevated the yield to 72.29 U/mL and 68.88 U/mL, respectively. Ultimately, a 10.79-fold increase in ZENM production was achieved in shake flask cultures compared to the initial strain. This research establishes a solid foundation for advancing the industrial application of ZENM.