Mutation, food-grade expression, and characterization of a lactonase for zearalenone degradation

Abstract

Zearalenone (ZEN) is a mycotoxin that causes serious threats to human health. People are exposed to ZEN contamination externally and internally through many ways, while environmental-friendly strategies for efficient elimination of ZEN are urgently needed worldwide. Previous studies revealed that the lactonase Zhd101 from Clonostachys rosea can hydrolyze ZEN to low toxicity compounds. In this work, the enzyme Zhd101 was conducted with combinational mutations to enhance its application properties. The optimal mutant (V153H-V158F), named Zhd101.1, was selected and introduced into the food-grade recombinant yeast strain Kluyveromyces lactis GG799(pKLAC1-Zhd101.1), followed by induced expression and secretion into the supernatant. The enzymatic properties of this mutant were extensively examined, revealing a 1.1-fold increase in specific activity, as well as improved thermostability and pH stability, compared to the wild-type enzyme. The ZEN degradation tests and the reaction parameters optimization were carried out in both solutions and the ZEN-contaminated corns, using the fermentation supernatants of the food-grade yeast strain. Results showed that the degradation rates for ZEN by fermentation supernatants reached 96.9% under optimal reaction conditions and 74.6% in corn samples, respectively. These new results are a useful reference to zearalenone biodegradation technologies and indicated that the mutant enzyme Zhd101.1 has potential to be used in food and feed industries. KEY POINTS: • Mutated lactonase showed 1.1-fold activity, better pH stability than the wild type. • The strain K. lactis GG799(pKLAC1-Zhd101.1) and the mutant Zhd101.1 are food-grade. • ZEN degradation rates by supernatants reached 96.9% in solution and 74.6% in corns.

Keywords: Degradation; Enzyme mutation; Food-grade; Kluyveromyces lactis; Zearalenone.