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 Km, Vmax 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.
Highlights
Zearalenone (ZEN) is a nonsteroidal estrogenic mycotoxin which was first isolated by Stob et al in 1962 and its structure was determined by Urry (Perry et al 1970; Urry et al 1966)
Construction of expression plasmids Total of 196 codons of ZENC were optimized according to the codon usage of P. pastoris
The deduced amino acid sequence of ZENC was analyzed by using BLAST server and shows its highest identity (97%) with a hypothetical protein from N. tetrasperma, and identity of 29% with an experimentally-verified zearalenone hydrolase (ZHD101) from Clonostachys rosea
Summary
Zearalenone (ZEN) is a nonsteroidal estrogenic mycotoxin which was first isolated by Stob et al in 1962 and its structure was determined by Urry (Perry et al 1970; Urry et al 1966) It was initially used as a growth promoter in animal feed (Wilson et al 1972), but it is considered to have adverse effects, mainly on the reproduction of animals (Kleinova et al 2002). This mycotoxin is a secondary metabolite of Fusarium species including F. graminearum, F. culmorum, F. equiseti, F. semitectum and F. crookwellense, F. graminearum. Contamination can be caused by improper storage of cereals
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