Identification of a Potent Enzyme for the Detoxification of Zearalenone.

The occurrence of mycotoxin zearalenone (ZEN) and its derivatives has been a severe global threat to food and animals. In addition to the chemical and physical degradation methods, a powerful biocatalyst is urgently required for the detoxification of ZEN. Here, an efficient ZEN-degrading lactonase from Gliocladium roseum, named ZENG, was identified for the first time. The recombinant ZENG exhibited the highest activity at pH 7.0 and 38 °C. In addition, the recombinant enzyme showed a high degrading performance toward ZEN and its toxic derivatives α-zearalenol (α-ZOL) and α-zearalanol (α-ZAL), with the specific activities as 315, 187, and 117 units/mg, respectively. To meet the industrial demands, attempts were also made to enhance the thermostability of ZENG using a structure-based modification. Three double-site mutants, including H134L/S136L, H134F/S136F, and H134I/S134I, in the position between the cap and core catalytic domain of ZENG were designed. Finally, the thermostability of both H134L/S136L and H134F/S136F displayed a significant improvement compared to the wild-type enzyme.

Expression, functional analysis and mutation of a novel neutral zearalenone-degrading enzyme.

The crops and grains were often contaminated by high level of mycotoxin zearalenone (ZEN). In order to remove ZEN and keep food safe, ZEN-degrading or detoxifying enzymes are urgently needed. Here, a newly identified lactonohydrolase responsible for the detoxification of ZEN, annotated as Zhd518, was expressed and characterized. Zhd518 showed 65% amino acid identity with Zhd101, which was widely studied for its ZEN-degrading ability. A detailed activity measurement method of ZEN-degrading enzyme was provided. Biochemical analysis indicated that the purified recombinant Zhd518 from E. coli exhibited a high activity against ZEN (207.0 U/mg), with the optimal temperature and pH of 40 °C and 8.0, respectively. The Zhd518 can degrade ZEN derivatives, and the specific activities against α-Zearalenol, ß-Zearalenol, α-Zearalanol and ß-Zearalanol were 23.0 U/mg, 64.7 U/mg, 119.8 U/mg and 66.5 U/mg, respectively. The active sites of Zhd518 were predicted by structure modeling and determined by mutation analysis. A point mutant N156H exhibited 3.3-fold activity against α-Zearalenol comparing to Zhd518. Zhd518 is the first reported neutral and the second characterized ZEN-degrading enzyme, which provides a new and more excellent candidate for ZEN detoxifying in food and feed industry.

Biological control of wood decay against fungal infection.

Wood (timber) is an important raw material for various purposes, and having biological composition it is susceptible to deterioration by various agents. The history of wood protection by impregnation with synthetic chemicals is almost two hundred years old. However, the ever-increasing public concern and the new environmental regulations on the use of chemicals have created the need for the development and the use of alternative methods for wood protection. Biological wood protection by antagonistic microbes alone or in combination with (bio)chemicals, is one of the most promising ways for the environmentally sound wood protection. The most effective biocontrol antagonists belong to genera Trichoderma, Gliocladium, Bacillus, Pseudomonas and Streptomyces. They compete for an ecological niche by consuming available nutrients as well as by secreting a spectrum of biochemicals effective against various fungal pathogens. The biochemicals include cell wall-degrading enzymes, siderophores, chelating iron and a wide variety of volatile and non-volatile antibiotics. In this review, the nature and the function of the antagonistic microbes in wood protection are discussed.

Cloning and expression analysis of a chitinase gene Crchi1 from the mycoparasitic fungus Clonostachys rosea (syn. Gliocladium roseum).

Clonostachys rosea (syn. Gliocladium roseum) is a well-known biocontrol agent and widely distributed around the world. In this study, an endochitinase gene Crchi1 was isolated from the mycoparasitic fungus C. rosea using the DNA walking strategy. The Crchi1 ORF is 1,746 bp long and interrupted by three introns. The cloned gene Crchi1 encodes 426 amino acid residues and shares a high degree of similarity with other chitinases from entomopathogenic and mycoparasitic fungi. Several putative binding sites for transcriptional regulation of Crchi1 in response to carbon (5'-SYGGRG-3') and nitrogen (5'-GATA-3') were identified in the upstream of Crchi1. Expression of Crchi1 gene in different carbon sources was analyzed using real-time PCR (RT-PCR). We found that the Crchi1 expression was suppressed by glucose but strongly stimulated by chitin or solubilized components of the cell wall from Rhizoctonia solani. Phylogenetic analysis of chitinases from entomopathogenic and mycoparasitic fungi suggests that these chitinases have probably evolved from a common ancestor.

Role of zearalenone lactonase in protection of Gliocladium roseum from fungitoxic effects of the mycotoxin zearalenone.

Zearalenone is a mycotoxin with estrogenic effects on mammals that is produced by several species of Fusarium. We found that zearalenone and its derivatives inhibit the growth of filamentous fungi on solid media at concentrations of < or =10 microg/ml. The fungitoxic effect declined in the order zearalenone > alpha-zearalenol > beta-zearalenol. The mycoparasitic fungus Gliocladium roseum produces a zearalenone-specific lactonase which catalyzes the hydrolysis of zearalenone, followed by a spontaneous decarboxylation. The growth of G. roseum was not inhibited by zearalenone, and the lactonase may protect G. roseum from the toxic effects of this mycotoxin. We inactivated zes2, the gene encoding zearalenone lactonase in G. roseum, by inserting a hygromycin resistance cassette into the coding sequence of the gene by means of Agrobacterium tumefaciens-mediated genetic transformation. The zes2 disruption mutants could not hydrolyze the lactone bond of zearalenone and were more sensitive to zearalenone. These data are consistent with a hypothesis that resorcylic acid lactones exemplified by zearalenone act to reduce growth competition by preventing competing fungi from colonizing substrates occupied by zearalenone producers and suggest that they may play a role in fungal defense against mycoparasites.

Efficacy of microorganisms antagonistic to Rhizoctonia cerealis and their cell wall degrading enzymatic activities.

The effect of Trichoderma atroviride, T. harzianum, T. longibrachiatum, Clonostachys rosea and Bacillus subtilis isolates applied to wheat seeds against Rhizoctonia cerealis disease of seedlings was investigated under controlled greenhouse conditions. Most Trichoderma isolates significantly reduced the incidence of disease compared with the infected control. Bacillus subtilis was also effective against sharp eyespot, although less active than Trichoderma spp. Interactions between the antagonistic microorganisms and the cereal pathogenic fungus in dual culture experiments on agar growth medium were also studied. Almost all tested antagonists showed competitive activity against R. cerealis: inhibition of its mycelial growth and hyphal interaction. The production of extracellular beta-N-acetylhexosaminidase, chitin 1,4-beta-chitobiosidase, glucan 1,3-beta-glucosidase and protease activity by the tested microorganisms in the presence of cell walls of R. cerealis was then determined. All isolates showed glucosaminidase and chitobiosidase activity. They also produced glucosidase activity, except B. subtilis, whereas only C. rosea, B. subtilis and one isolate of T. harzianum showed detectable levels of protease activity.

A novel lactonohydrolase responsible for the detoxification of zearalenone: enzyme purification and gene cloning.

Zearalenone (ZEN) is converted into a far less oestrogenic product by incubation with Clonostachys rosea IFO 7063. An alkaline hydrolase responsible for the detoxification was purified to homogeneity from the fungus by a combination of salt precipitation and column chromatography methods. The purified enzyme was homodimeric with a subunit molecular mass of 30 kDa and contained an intra-subunit disulphide bridge. On the basis of the internal peptide sequences of the purified protein, we cloned the entire coding region of the gene (designated as zhd101) by PCR techniques. The ZEN degradation activity was detected in heterologous hosts (Schizosaccharomyces pombe and Escherichia coli) carrying the cloned gene. Zhd101 could be a promising genetic resource for in planta detoxification of the mycotoxin in important crops.

Enzyme assisted ensiling of alfalfa with enzymes by solid substrate fermentation.

A crude enzyme preparation, obtained by solid substrate fermentation (SSF) with a Gliocladium spp. and added at the 5% level to wilted or non-wilted alfalfa, improved the fermentation characteristics and stability of alfalfa silages as effectively as commercial preparations, Novo-Nordisk Celluclast 1.5 L and Viscozyme 120 L, applied at the 0.025% level. The effective dose of the crude enzyme costs about one-fourth of the cost of the commercial enzymes.