Characteristics of a Novel Zearalenone Lactone Hydrolase ZHRnZ and Its Thermostability Modification

Abstract

Zearalenone (ZEN) is a toxic secondary metabolite produced by the Fusarium fungi, which widely contaminates grains, food, and feed, causing health hazards for humans and animals. Therefore, it is essential to find effective ZEN detoxification methods. Enzymatic degradation of ZEN is believed to be an eco-friendly detoxification strategy, specifically thermostable ZEN degradation enzymes are needed in the food and feed industry. In this study, a novel ZEN lactone hydrolase ZHRnZ from Rosellinia necatrix was discovered using bioinformatic and molecular docking technology. The recombinant ZHRnZ showed the best activity at pH 9.0 and 45 °C with more than 90% degradation for ZEN, α-zearalenol (α-ZOL), β-zearalenol (β-ZOL) and α-zearalanol (α-ZAL) after incubation for 15 min. We obtained 10 mutants with improved thermostability by single point mutation technology. Among them, mutants E122Q and E122R showed the best performance, which retained more than 30% of their initial activity at 50 °C for 2 min, and approximately 10% of their initial activity at 60 °C for 1 min. The enzymatic kinetic study showed that the catalytic efficiency of E122R was 1.3 times higher than that of the wild-type (WT). Comprehensive consideration suggests that mutant E122R is a promising hydrolase to detoxify ZEN in food and feed.

Keywords: lactone hydrolase; mutation; thermostability; zearalenone.

Figure 1

Enzymatic degradation of ZEN by lactone hydrolase.

Figure 2

(A) Protein sequence alignment between ZHRnZ and ZHD101. The green triangles represent the catalytic triad of ZHRnZ. (B) The simulative crystal structure of a subunit of ZHRnZ. Blue, yellow, red, and cyan represent the α-helices 4–7, respectively. Pink represents the catalytic triad. Orange represents the mutation site.

Figure 3

Results of molecular docking between ZHRnZ and ZEN. (A) The molecular docking box. Docking site: x = −16.747, y = 17.206, z = 6.303. radius: 11 Å. (B) The interaction between ZEN and ZHRnZ. Inside the dotted circle is the catalytic pocket of ZHRnZ. (C) Substrate–enzyme interaction networks of ZHRnZ. The red labels represent the amino acid residues involved in forming hydrogen bonds. The blue label represents the amino acid residues involved in forming π–π bonds. The black labels represent other amino acid residues involved in forming hydrophobic forces. (D) 2D display of interaction force of substrate-enzyme.

Figure 4

(A) The map of recombinant plasmid pET28a-ZHRnZ. The blue part represents the optimized ZHRnZ gene sequence. (B) SDS-PAGE analysis of the recombinant ZHRnZ. Lane M represents the protein marker (116.0, 66.2, 45.0, 35.0, 25.0, 18.4 and 14.4 kDa). (C) The degradation rate of different concentrations of ZEN using recombinant ZHRnZ. (D) The degradation rate of ZEN and its derivatives using recombinant ZHRnZ.

Figure 5

Enzymatic properties of recombinant ZHRnZ. (A) The optimal pH of recombinant ZHRnZ for the degradation of ZEN. (B) The optimal temperature of recombinant ZHRnZ for the degradation of ZEN. (C) The pH resistance of recombinant ZHRnZ for the degradation of ZEN. (D) The thermostability of recombinant ZHRnZ for the degradation of ZEN.

Figure 6

The RMSF results of amino acid residues in ZHRnZ.

Figure 7

The characteristics of the mutants. (A) SDS-PAGE analysis of mutants. Lane M represents the protein ladder (116.0, 66.2, 45.0, 35.0, 25.0, 18.4, and 14.4 kDa); Lanes 1–10 were mutants E122I, E122H, E122M, E122Q, E122R, E122L, E122K, E122S, E122W, and E122T, respectively. (B) The effect of WT and mutants on the degradation of ZEN. (C) The residual activity of WT and mutants at 40 °C for 5 min. (D) The residual activity of WT and mutants at 40 °C for 15 min. (E) The residual activity of WT and mutants at 50 °C for 2 min. (F) The residual activity of mutants E122Q, E122M, E122S, and E122R at 60 °C for 1 min.