Characterization, expression and application of a zearalenone degrading enzyme from Neurospora crassa

Abstract

A gene named zenc, encoding a zearalenone lactonase from Neurospora crassa, was over-expressed in Pichia pastoris. The zenc gene is 888-bp in length, encoding a 295-residue polypeptide. Purified ZENC has maximal activity at pH 8.0 and 45 °C, and is highly stable at pH 6.0-8.0 for 1 h at 37 °C. The activity of the secreted enzyme in shaken-flask fermentation was 40.0 U/ml. A high-density fermentation of the ZENC-producing recombinant strain was performed in a 30-l fermenter and the maximal enzyme activity reached 290.6 U/ml. The K_m, V_max and specific activity toward zearalenone are 38.63 μM, 23.8 μM/s/mg and 530.4 U/mg, respectively. ZENC can resist metal ions and inhibitors to some extent. We applied the enzyme into three different kinds of animal feed. On addition of ZENC (800 U) to distillers dried grains with solubles (DDGS), maize by-products and corn bran (25 g), the concentration of zearalenone was reduced by 70.9%, 88.9% and 94.7% respectively. All these properties of ZENC are promising for applications in the animal feed and food industries.

Keywords: Animal feed; High-density fermentation; Maize; Pichia pastoris; Zearalenone degradation.

Fig. 1

a Electrophoresis analysis of expression plasmid. lane M: marker; lane 1: recombined plasmid; lane 2: recombined plasmid was digested by EcoRI and NotI; lane 3: pPIC9K was digested by EcoRI and NotI. b Electrophoresis analysis of PCR. lane M: marker; lane 1: PCR of P. pastoris; lane 2: PCR of recombinant P. pastoris. c Alignment of the deduced amino acid sequence of ZENC (1, upper line) with 2-(acetamidomethylene) succinate hydrolase from Mesorhizobium japonicum (2, lower line). Identical (.) are shown. Residues that comprise the catalytic triad of a serine hydrolase family are boxed

Fig. 2

Effect of temperature and pH on the enzyme activity of recombinant ZENC. a Effect of pH on the enzyme activity. b pH stability of recombinant enzyme. c Effect of temperature on the enzyme activity. d Thermostability of recombinant enzyme. Error bars represent the standard devision of values and every case was carried out three experiments

Fig. 3

High-density fermentation of recombinant P. pastoris expressing ZENC in a 30-l fermenter. a Wet weight of cells and enzyme activity. b SDS-PAGE analysis of culture supernatant from the 30-l fermenter at different periods of incubation time; Lanes 1–9, sample supernatants from culture induced with methanol for 12, 24, 36, 48, 60, 72, 84, 96 and 108 h, respectively

Fig. 4

Zearalenone (ZEN) degradation in different substrates on addition of ZENC. a ZEN degradation in DDGS. b ZEN degradation in maize by-products. c ZEN degradation in corn bran