Patulin Detoxification by Recombinant Manganese Peroxidase from Moniliophthora roreri Expressed by Pichia pastoris

Abstract

The fungal secondary metabolite patulin is a mycotoxin widespread in foods and beverages which poses a serious threat to human health. However, no enzyme was known to be able to degrade this mycotoxin. For the first time, we discovered that a manganese peroxidase (MrMnP) from Moniliophthora roreri can efficiently degrade patulin. The MrMnP gene was cloned into pPICZα(A) and then the recombinant plasmid was transformed into Pichia pastoris X-33. The recombinant strain produced extracellular manganese peroxidase with an activity of up to 3659.5 U/L. The manganese peroxidase MrMnP was able to rapidly degrade patulin, with hydroascladiol appearing as a main degradation product. Five mg/L of pure patulin were completely degraded within 5 h. Moreover, up to 95% of the toxin was eliminated in a simulated patulin-contaminated apple juice after 24 h. Using Escherichia coli as a model, it was demonstrated that the deconstruction of patulin led to detoxification. Collectively, these traits make MrMnP an intriguing candidate useful in enzymatic detoxification of patulin in foods and beverages.

Keywords: apple juice; detoxification; manganese peroxidase; mycotoxin; patulin.

Figure 1

Schematic diagram showing the use of a recombinant MrMnP expressed in P. pastoris to degrade and detoxify patulin, a mycotoxin commonly discovered in fruits.

Figure 2

Expression of MrMnP in P. pastoris. (A) The plasmid map of pPICZα(A)-MrMnP. (B) SDS-PAGE analysis of the MrMnP enzyme recombinantly produced in P. pastoris. The arrow indicates the recombinant MrMnP protein. Lane M: protein molecular mass marker; 1: MrMnP protein recombinantly produced in P. pastoris.

Figure 3

Effects of the buffer components on patulin transformation. The reactions were carried out by incubating 0.5 U/mL of MrMnP with 5 mg/L of patulin in one of the buffers containing malonate, acetate, oxalate, citrate, lactate, phosphate, MES, and HEPES at 30 °C for 24 h. The data in the picture is the average ± standard deviation, *** p < 0.001, One Way anova test.

Figure 4

Mn²⁺ played an important role in degrading patulin. The reactions were carried out by incubating 0.5 U/mL of MrMnP with 5 mg/L of patulin in the acetate or malonate buffer in absence or presence of Mn²⁺ at 30 °C for 24 h. Then the products were analyzed by HPLC.

Figure 5

MrMnP-catalyzed degradation of patulin led to detoxification. (A) Patulin was toxic to E. coli as demonstrated by retarded growth of the bacterium. A series of concentrations (1, 10, 50, and 100 mg/L) of patulin were added to equal amounts of E. coli and the culture was continued at 37 °C for 12 h. (B) MrMnP-catalyzed degradation of patulin alleviated the retarding effect of patulin on E. coli. Patulin (1, 10, 50, and 100 mg/L) was first treated with 0.5 U/mL of MrMnP at 30 °C for 24 h and then added to E coli.

Figure 6

Identification of hydroascladiol as one major transformation product of patulin. PAT was incubated with 0.5 U/mL of MrMnP in 50 mM malonate buffer (pH 5.0) supplemented with 1 mM MnSO₄ and 0.1 mM H₂O₂ and the reaction was carried out at 30 °C for 8 h. The degradation products were analyzed by HPLC–MS/MS.

Figure 7

MrMnP efficiently catalyzed degradation of patulin in a simulated patulin-contaminated apple juice. The reactions were carried out by incubating 0.5 U/mL of MrMnP with 5 mg/L in absence (or presence) of apple juice. The samples were periodically taken out for HPLC analysis. The data in the picture is the average ± standard deviation, *** p < 0.001, ** p < 0.01, * p < 0.05, One Way anova test.