Family Report for: Zearalenone-hydrolase

Lactonohydrolase ZHD can detoxify oestrogenic mycotoxin zearalenone and zearalenols through hydrolysis and decarboxylation. The detail mechanism, especially the role of Trp183, which interacts with substrate through p-pi interaction and one hydrogen bond, is still unknown. The Trp183 mutants abolished activity to ZEN, alpha-ZOL and beta-ZOL, except that W183F mutant retained about 40% activity against alpha-ZOL. In two W183F-reactant complex structures the reactants still bind at the active position and it suggested that this p-pi interaction takes responsible for the reactants recognization and allocation. Further, the ZHD-productant complex structures showed that the resorcinol ring of hydrolysed alpha-ZOL and hydrolysed beta-ZOL move a distance of one ring as compare to the resorcinol ring of reactant alpha-ZOL and beta-ZOL. The same movement also found in comparison of hydrolysed ZEN and ZEN. In the structure of W183F complex with hydrolysed alpha-ZOL the resorcinol ring of hydrolysed alpha-ZOL doesn't move as compare to the resorcinol ring of reactant alpha-ZOL. It suggested the Trp183 coordinated hydrogen bond takes responsible for the movement of the hydrolysed product. These functional and structural results suggested that Trp183 is essential for ZHD detoxifying zearalenone and zearalenols.

Zearalenone hydrolase (ZHD) is the only reported alpha/beta-hydrolase that can detoxify zearalenone (ZEN). ZHD has demonstrated its potential as a treatment for ZEN contamination that will not result in damage to cereal crops. Recent researches have shown that the V153H mutant ZHD increased the specific activity against alpha-ZOL, but decreased its specific activity to beta-ZOL. To understand whyV153H mutation showed catalytic specificity for alpha-ZOL, four molecular dynamics simulations combining with protein network analysis for wild type ZHD alpha-ZOL, ZHD beta-ZOL, V153H alpha-ZOL, and V153H beta-ZOL complexes were performed using Gromacs software. Our theoretical results indicated that the V153H mutant could cause a conformational switch at the cap domain (residues Gly161(-)Thr190) to affect the relative position catalytic residue (H242). Protein network analysis illustrated that the V153H mutation enhanced the communication with the whole protein and residues with high betweenness in the four complexes, which were primarily assembled in the cap domain and residues Met241 to Tyr245 regions. In addition, the existence of alpha-ZOL binding to V153H mutation enlarged the distance from the OAE atom in alpha-ZOL to the NE2 atom in His242, which prompted the side chain of H242 to the position with catalytic activity, thereby increasing the activity of V153H on the alpha-ZOL. Furthermore, alpha-ZOL could easily form a right attack angle and attack distance in the ZHD and alpha-ZOL complex to guarantee catalytic reaction. The alanine scanning results indicated that modifications of the residues in the cap domain produced significant changes in the binding affinity for alpha-ZOL and beta-ZOL. Our results may provide useful theoretical evidence for the mechanism underlying the catalytic specificity of ZHD.

Zearalenone (ZEA) is an oestrogenic mycotoxin produced by several Fusarium species, and it frequently contaminates cereals used for food or animal feed. A ZEA-lactonase of Gliocladium roseum was previously described to hydrolyse ZEA to an unstable intermediate, which spontaneously decarboxylates to non-oestrogenic, decarboxylated hydrolysed ZEA (DHZEN). We expressed a codon-optimised version of the ZEA-lactonase (ZHD101) gene of G. roseum MA 918 with a secretion leader in Pichia pastoris and purified the recombinant enzyme from culture supernatant by His-tag mediated affinity chromatography. After incubation of the enzyme with ZEA, we detected the previously elusive primary reaction product hydrolysed ZEA (HZEN) by liquid chromatography tandem mass spectrometry, purified it by preparative high-performance liquid chromatography, and confirmed its postulated structure ((E)-2,4-dihydroxy-6-(10-hydroxy-6-oxo-1-undecen-1-yl)benzoic acid) by nuclear magnetic resonance techniques. Spontaneous decarboxylation to DHZEN ((E)-1-(3,5-dihydroxy-phenyl)-10-hydroxy-1-undecen-6-one), but not to a previously reported isomer, was observed. Biomass resuspensions of G. roseum strains MA 918 and the strains used for previous work, NBRC 7063 and ATCC 8684, all converted ZEA to HZEN, DHZEN, and further unknown metabolites. We studied partitioning of HZEN and DHZEN between aqueous phases and organic solvents, and found that HZEN did not partition into chloroform as extraction solvent, under the conditions used by previous authors. In contrast, extraction with ethyl acetate at pH 2.0 was suitable for simultaneous extraction of HZEN and DHZEN. The detection of HZEN and its availability as an analytical standard may assist further work towards possible application of ZEA-lactonase (e.g. determining kinetic parameters) for detoxification of ZEA.

Lactonohydrolase ZHD can detoxify oestrogenic mycotoxin zearalenone and zearalenols through hydrolysis and decarboxylation. The detail mechanism, especially the role of Trp183, which interacts with substrate through p-pi interaction and one hydrogen bond, is still unknown. The Trp183 mutants abolished activity to ZEN, alpha-ZOL and beta-ZOL, except that W183F mutant retained about 40% activity against alpha-ZOL. In two W183F-reactant complex structures the reactants still bind at the active position and it suggested that this p-pi interaction takes responsible for the reactants recognization and allocation. Further, the ZHD-productant complex structures showed that the resorcinol ring of hydrolysed alpha-ZOL and hydrolysed beta-ZOL move a distance of one ring as compare to the resorcinol ring of reactant alpha-ZOL and beta-ZOL. The same movement also found in comparison of hydrolysed ZEN and ZEN. In the structure of W183F complex with hydrolysed alpha-ZOL the resorcinol ring of hydrolysed alpha-ZOL doesn't move as compare to the resorcinol ring of reactant alpha-ZOL. It suggested the Trp183 coordinated hydrogen bond takes responsible for the movement of the hydrolysed product. These functional and structural results suggested that Trp183 is essential for ZHD detoxifying zearalenone and zearalenols.

The danger of zearalenone (ZEN) as an endocrine disruptor to humans and the environment has aroused increasing attention. In this study, we implemented the quantum mechanics/molecular mechanics (QM/MM) method to investigate the degradation mechanism of ZEN hydrolase (RmZHD) toward ZEN at the atomic level. The degradation process involves two concerted reaction pathways, where the active site contains a Ser-His-Glu triplet as a proton donor. With the Boltzmann-weighted average potential barriers of 18.1 and 21.5 kcal/mol, the process undergoes proton transfer and nucleophilic-substituted ring opening to form a hydroxyl product. Non-covalent interaction analyses elucidated hydrogen bonding between key amino acids with ZEN. The electrostatic influence analysis of 16 amino acids proposes residues Asp34 and His128 as the possible mutation target for future mutation design of enzyme RmZHD. An in-depth investigation of the protein environment of RmZHD can improve the bioremediation efficiency of endocrine disrupting chemicals.

Zearalenone hydrolase (ZHD) is the only reported alpha/beta-hydrolase that can detoxify zearalenone (ZEN). ZHD has demonstrated its potential as a treatment for ZEN contamination that will not result in damage to cereal crops. Recent researches have shown that the V153H mutant ZHD increased the specific activity against alpha-ZOL, but decreased its specific activity to beta-ZOL. To understand whyV153H mutation showed catalytic specificity for alpha-ZOL, four molecular dynamics simulations combining with protein network analysis for wild type ZHD alpha-ZOL, ZHD beta-ZOL, V153H alpha-ZOL, and V153H beta-ZOL complexes were performed using Gromacs software. Our theoretical results indicated that the V153H mutant could cause a conformational switch at the cap domain (residues Gly161(-)Thr190) to affect the relative position catalytic residue (H242). Protein network analysis illustrated that the V153H mutation enhanced the communication with the whole protein and residues with high betweenness in the four complexes, which were primarily assembled in the cap domain and residues Met241 to Tyr245 regions. In addition, the existence of alpha-ZOL binding to V153H mutation enlarged the distance from the OAE atom in alpha-ZOL to the NE2 atom in His242, which prompted the side chain of H242 to the position with catalytic activity, thereby increasing the activity of V153H on the alpha-ZOL. Furthermore, alpha-ZOL could easily form a right attack angle and attack distance in the ZHD and alpha-ZOL complex to guarantee catalytic reaction. The alanine scanning results indicated that modifications of the residues in the cap domain produced significant changes in the binding affinity for alpha-ZOL and beta-ZOL. Our results may provide useful theoretical evidence for the mechanism underlying the catalytic specificity of ZHD.

Zearalenone (ZEN) is a mycotoxin which causes huge economic losses in the food and animal feed industries. The lactonase ZHD101 from Clonostachys rosea, which catalyzes the hydrolytic degradation of ZEN, is the only known ZEN-detoxifying enzyme. Here, a protein homologous to ZHD101, denoted CbZHD, from Cladophialophora batiana was expressed and characterized. Sequence alignment indicates that CbZHD possesses the same catalytic triad and ZEN-interacting residues as found in ZHD101. CbZHD exhibits optimal enzyme activity at 35 degrees C and pH 8, and is sensitive to heat treatment. The crystal structure of apo CbZHD was determined to 1.75 A resolution. The active-site compositions of CbZHD and ZHD101 were analyzed.

Zearalenone hydrolase (ZHD) is an alpha/beta-hydrolase that detoxifies and degrades the lactone zearalenone (ZEN), a naturally occurring oestrogenic mycotoxin that contaminates crops. Several apoenzyme and enzyme-substrate complex structures have been reported in the resolution range 2.4-2.6 A. However, the properties and mechanism of this enzyme are not yet fully understood. Here, a 1.60 A resolution structure of a ZHD-product complex is reported which was determined from a C-terminally His6-tagged ZHD crystal soaked with 2 mM ZEN for 30 min. It shows that after the lactone-bond cleavage, the phenol-ring region moves closer to residues Leu132, Tyr187 and Pro188, while the lactone-ring region barely moves. Comparisons of the ZHD-substrate and ZHD-product structures show that the hydrophilic interactions change, especially Trp183 N1, which shifts from contacting O2 to O12', suggesting that Trp183 is responsible for the unidirectional translational movement of the phenol ring. This structure provides information on the final stage of the catalytic mechanism of zearalenone hydrolysis.

Zearalenone (ZEN), mainly produced by Fusarium species, is an estrogenic mycotoxin which causes reproductive disorders in livestock. In this study, we described a simple and rapid method for screening of ZEN-degrading bacteria by esterase activity assay. Soil bacteria strains were first tested for their esterase activities, then active strains were further evaluated for their ZEN-degrading potentials. A bacterial strain named Bacillus pumilus ES-21 was detected to be able to eliminate ZEN in the culture medium. ZEN degradation conditions were optimized through response surface methodology and the result showed that the degradation rate of ZEN by Bacillus pumilus ES-21 was up to 95.7% at the ZEN concentration of 17.9 mug/ml within 24 h. One of the degradation product was proposed to be 1-(3,5-dihydroxyphenyl)-6'-hydroxy-l'-undecen-l0'-one according to LC-TOF-MS/MS analysis. This study provided a strategy for the isolation of ZEN degrading microbes and a promising degrading strain.

Zearalenone (ZEA) is an oestrogenic mycotoxin produced by several Fusarium species, and it frequently contaminates cereals used for food or animal feed. A ZEA-lactonase of Gliocladium roseum was previously described to hydrolyse ZEA to an unstable intermediate, which spontaneously decarboxylates to non-oestrogenic, decarboxylated hydrolysed ZEA (DHZEN). We expressed a codon-optimised version of the ZEA-lactonase (ZHD101) gene of G. roseum MA 918 with a secretion leader in Pichia pastoris and purified the recombinant enzyme from culture supernatant by His-tag mediated affinity chromatography. After incubation of the enzyme with ZEA, we detected the previously elusive primary reaction product hydrolysed ZEA (HZEN) by liquid chromatography tandem mass spectrometry, purified it by preparative high-performance liquid chromatography, and confirmed its postulated structure ((E)-2,4-dihydroxy-6-(10-hydroxy-6-oxo-1-undecen-1-yl)benzoic acid) by nuclear magnetic resonance techniques. Spontaneous decarboxylation to DHZEN ((E)-1-(3,5-dihydroxy-phenyl)-10-hydroxy-1-undecen-6-one), but not to a previously reported isomer, was observed. Biomass resuspensions of G. roseum strains MA 918 and the strains used for previous work, NBRC 7063 and ATCC 8684, all converted ZEA to HZEN, DHZEN, and further unknown metabolites. We studied partitioning of HZEN and DHZEN between aqueous phases and organic solvents, and found that HZEN did not partition into chloroform as extraction solvent, under the conditions used by previous authors. In contrast, extraction with ethyl acetate at pH 2.0 was suitable for simultaneous extraction of HZEN and DHZEN. The detection of HZEN and its availability as an analytical standard may assist further work towards possible application of ZEA-lactonase (e.g. determining kinetic parameters) for detoxification of ZEA.

The mycotoxin zearalenone (ZEN) can be degraded by a lactone hydrolase ZHD, which was derived from Gliocladium roseum. Here, based on the native ZHD encoding gene zhd101, a codon optimized zhd gene was synthesized, which was used for high expression of ZHD in Pichia pastoris GS115. Meanwhile, to further improve the expression of recombinant ZHD, the plasmids containing 1 to 4 copies of the zhd expression cassette were constructed, respectively, using the biobrick method. The protein expression in the recombinant P. pastoris X3c, which was transformed with the plasmid containing 3 copies of zhd expression cassette, was the highest. In addition, the enzymatic activity of ZHD against ZEN was defined for the first time based on a standard curve of peak area vs ZEN concentration. The ZEN degradation activity of ZHD from shake flask fermentation was calculated as 22.5U/mL with the specific activity of 4976.5U/mg. Furthermore, the high-density fermentation of P. pastoris X3c strain was also performed in 5L fermenter. The maximum enzyme activity of the supernatant was 150.1U/mL, which were 6.7-fold higher than that of the shake flask fermentation.

The fungus Clonostachys rosea is antagonistic against plant pathogens, including Fusarium graminearum, which produces the oestrogenic mycotoxin zearalenone (ZEA). ZEA inhibits other fungi, and C. rosea can detoxify ZEA through the enzyme zearalenone lactonohydrolase (ZHD101). As the relevance of ZEA detoxification for biocontrol is unknown, we studied regulation and function of ZHD101 in C. rosea. Quantitative reverse-transcription PCR revealed zhd101 gene expression in all conditions studied and demonstrated dose-dependent induction by ZEA. Known inducers of the Polyketide Synthase pathway did not induce zhd101 expression, suggesting specificity of the enzyme towards ZEA. To assess the role of ZHD101 during biocontrol interactions, we generated two Deltazhd101 mutants incapable of ZEA-detoxification and confirmed their defect in degrading ZEA by HPLC. The Deltazhd101 mutants displayed a lower in vitro ability to inhibit growth of the ZEA-producing F. graminearum (strain 1104-14) compared to the wild type. In contrast, all three C. rosea strains equally inhibited growth of the F. graminearum mutant (DeltaPKS4), which is impaired in ZEA-production. Furthermore, the Deltazhd101 mutants failed to protect wheat seedlings against foot rot caused by the ZEA-producing F. graminearum. These data show that ZEA detoxification by ZHD101 is important for the biocontrol ability of C. rosea against F. graminearum.

The mycotoxin zearalenone has been contaminating maize and other grains. It can be hydrolyzed and inactivated by the lactonase ZHD, which belongs to the alpha/beta-hydrolase family. Besides the catalytic core domain, the enzyme comprises an alpha-helical cap domain. Zearalenone differs from other quorum-sensing lactones in its chemical structure. As revealed by the complex structure, the substrate binds into a deep pocket between the core and cap domains, adjacent to the catalytic triad Ser102-His242-Glu126. The enzyme-substrate interactions include three direct hydrogen bonds and several nonpolar contacts. In particular, the Trp183 side chain is engaged in both hydrogen bonding and T-stacking interactions with the benzoate ring. The central role of Trp183 in substrate binding was verified by the mutants W183A, W183H and W183F. Several mutants were also produced to investigate the roles of nearby amino-acid residues. Interestingly, mutants that destabilize the dimer had adverse functional effects on ZHD.

BACKGROUND: Zearalenone is a mycotoxin produced by several species of Fusarium genus, most notably Fusarium graminearum and Fusarium culmorum. This resorcylic acid lactone is one of the most important toxins causing serious animal and human diseases. For over two decades it has been known that the mycoparasitic fungus Clonostachys rosea (synonym: Gliocladium roseum, teleomorph: Bionectria ochroleuca) can detoxify zearalenone, however no such attributes have been described within the Trichoderma genus.
RESULTS: We screened for the presence of zearalenone lactonohydrolase homologs in isolates of Clonostachys and Trichoderma genera. We report first finding of expressed zearalenone lactonohydrolase in Trichoderma aggressivum. For three isolates (T. aggressivum, C. rosea and Clonostachys catenulatum isolates), we were able to reconstruct full coding sequence and verify the biotransformation ability potential. Additionally, we assessed progression of the detoxification process (in terms of transcript accumulation and mycotoxin decomposition in vitro).In silico, search for origins of zearalenone lactonohydrolase activity in model fungal and bacterial genomes has shown that zearalenone lactonohydrolase homologs form a monophyletic fungal clade among the a/b hydrolase superfamily representatives. We corroborated the finding of functional enzyme homologs by investigating the functional sites (active site pocket with postulated, noncanonical Ser-Glu-His catalytic triad) conserved in both multiple sequence alignment and in homology-based structural models.
CONCLUSIONS: Our research shows the first finding of a functional zearalenone lactonohydrolase in mycoparasitic Trichoderma aggressivum (an activity earlier characterised in the Clonostachys rosea strains). The supporting evidence for presence and activity of functional enzyme homologs is based on the chemical analyses, gene expression patterns, homology models showing conservation of key structural features and marked reduction of zearalenone content in cultured samples (containing both medium and mycelium). Our findings also show divergent strategies of zearalenone biotransformation ability (rapid induced expression and detoxification vs. gradual detoxification) present in several members of Hypocreales order (Trichoderma and Clonostachys genera). The potential for lactonhydrolase activity directed towards zearalenone and/or similar compounds is likely ancient, with homologs present in several divergent filamentous fungi among both Sordariomycetes (Bionectria sp., Trichoderma sp., Apiospora montagnei) and Leotiomycetes (Marssonina brunnea f. sp. 'multigermtubi').

Maize is subject to ear rot caused by toxigenic Aspergillus and Fusarium species, resulting in contamination with aflatoxins, fumonisins, trichothecenes, and zearalenone (ZEN). The trichothecene group and ZEN mycotoxins are produced by the cereal pathogen Fusarium graminearum. A transgenic detoxification system for the elimination of ZEN was previously developed using an egfp::zhd101 gene (gfzhd101), encoding an enhanced green fluorescent protein fused to a ZEN-degrading enzyme. In this study, we produced a transgenic maize line expressing an intact copy of gfzhd101 and examined the feasibility of transgene-mediated detoxification in the kernels. ZEN-degrading activity has been detected in transgenic kernels during seed maturation (for a period of 6 weeks after pollination). The level of detoxification activity was unaltered after an additional storage period of 16 weeks at 6 degrees C. When the seeds were artificially contaminated by immersion in a ZEN solution for 48 h at 28 degrees C, the total amount of the mycotoxin in the transgenic seeds was uniformly reduced to less than 1/10 of that in the wild type. The ZEN in the transgenic maize kernels was also efficiently decontaminated under conditions of lower water activity (aw) and temperature; e.g., 16.9 microg of ZEN was removed per gram of seed within 48 h at an aw of 0.90 at 20 degrees C. F. graminearum infection assays demonstrated an absence of ZEN in the transgenic maize seeds, while the mycotoxin accumulated in wild-type kernels under the same conditions. Transgene-mediated detoxification may offer simple solutions to the problems of mycotoxin contamination in maize.

Zearalenone (ZEN) is an estrogenic mycotoxin produced by the necrotrophic cereal pathogen Fusarium graminearum. This mycotoxin is detoxified by ZHD101, a lactonohydrolase from Clonostachys rosea, or EGFP:ZHD101, its fusion to the C-terminus of an enhanced green fluorescence protein. We previously showed that egfp:zhd101 is efficiently expressed in T(0) leaves of rice. In this study, we assessed the feasibility of in planta detoxification of the mycotoxin using progeny. When protein extract from T(1) leaves was incubated with ZEN, the amount of the toxin decreased significantly as measured by HPLC. ZEN degradation activity was also detected in vivo in transgenic T(2) seeds. These results suggest that zhd101 can be exploited as an efficient and cost-effective system for protection of important cereals that are more susceptible to the pathogen (e.g., wheat and maize) from contamination with the estrogenic mycotoxin.

Zearalenone (ZEA) is a polyketide mycotoxin produced by some species of Gibberella/Fusarium and causes hyperestrogenic syndrome in animals. ZEA occurs naturally in cereals infected by Gibberella zeae in temperate regions and threatens animal health. In this study, we report on a set of genes that participate in the biosynthesis of ZEA in G. zeae. Focusing on the non-reducing polyketide synthase (PKS) genes of the G. zeae genome, we demonstrated that PKS13 is required for ZEA production. Subsequent analyses revealed that a continuous, 50 kb segment of DNA carrying PKS13 consisted of three additional open reading frames that were coexpressed as a cluster during the condition for ZEA biosynthesis. These genes, in addition to PKS13, were essential for the ZEA biosynthesis. They include another PKS gene (PKS4) encoding a fungal reducing PKS; zearalenone biosynthesis gene 1 (ZEB1), which shows a high similarity to putative isoamyl alcohol oxidase genes; and ZEB2 whose deduced product carries a conserved, basic-region leucine zipper domain. ZEB1 is responsible for the chemical conversion of beta-zearalenonol (beta-ZOL) to ZEA in the biosynthetic pathway, and ZEB2 controls transcription of the cluster members. Transcription of these genes was strongly influenced by different culture conditions such as nutrient starvations and ambient pH. Furthermore, the same set of genes regulated by ZEB2 was dramatically repressed in the transgenic G. zeae strain with the deletion of PKS13 or PKS4 but not in the ZEB1 deletion strain, suggesting that ZEA or beta-ZOL may be involved in transcriptional activation of the gene cluster required for ZEA biosynthesis in G. zeae. This is the first published report on the molecular characterization of genes required for ZEA biosynthesis.

Zearalenone (ZEN), an estrogenic mycotoxin produced by several Fusarium species, is converted to a non-estrogenic product by a detoxifying enzyme of Clonostachys rosea. Previously, we investigated whether recombinant Saccharomyces cerevisiae carrying this detoxification gene, zhd101, can remove 2 microg ml(-1) of ZEN in a liquid culture. Although the transgenic yeasts eliminated most of the ZEN, they also converted a significant amount to a poor substrate, beta-zearalenol, which remained in the medium. In this study, we synthesized a codon-optimized zhd101 gene and investigated whether the transgenic yeast strain can overcome the problem of insufficient detoxification of ZEN. Importantly, within 48 h of incubation at 28 degrees C or 8 h of incubation at 37 degrees C, the transgenic yeasts completely eliminated 2 microg ml(-1) of ZEN in the medium without accumulating even a trace amount of beta-zearalenol. The result suggests that incomplete ZEN detoxification attributed to the action of an endogenous yeast beta-reductase can be overcome by simply increasing the expression of the detoxifying gene.

Zearalenone (ZEN) is converted to a nontoxic product by a lactonohydololase encoded by zhd101. An enhanced green fluorescent protein (EGFP) gene was fused to zhd101 (i.e., egfp::zhd101) and expressed in Escherichia coli. Both recombinant ZHD101 and EGFP::ZHD101 were purified to homogeneity and characterized. Maximal activity of ZHD101 toward ZEN was measured at approximately 37 to 45 degrees C and pH 10.5 (k(cat) at 30 degrees C, 0.51 s(-1)). The enzyme was irreversibly inactivated at pH values below 4.5 or by treatment with serine protease inhibitors. ZHD101 was also active against five ZEN cognates, although the efficiencies were generally low; e.g., the k(cat) was highest with zearalanone (1.5 s(-1)) and lowest with beta-zearalenol (0.075 s(-1)). EGFP::ZHD101 had properties similar to those of the individual proteins with regard to the EGFP fluorescence and lactonohydrolase activity. Fortuitously, EGFP::ZHD101 exhibited a good correlation between the fluorescence intensity and reaction velocity under various pH conditions. We therefore used egfp::zhd101 to visually monitor the lactonohydrolase activity in genetically modified organisms and evaluated the usefulness of zhd101 for in vivo detoxification of ZEN. While recombinant E. coli and transgenic rice calluses exhibited strong EGFP fluorescence and completely degraded ZEN in liquid media, recombinant Saccharomyces cerevisiae gave poor fluorescence and did not eliminate all the toxicity of the mycotoxin in the medium; i.e., the rest of ZEN was transformed into an unfavorable substrate, beta-zearalenol, by an as-yet-unidentified reductase and remained in the medium. Even so, as much as 75% of ZEN was detoxified by the yeast transformant, which is better than the detoxification system in which food-grade Lactobacillus strains are used (H. El-Nezami, N. Polychronaki, S. Salminen, and H. Mykkuane, Appl. Environ. Microbiol. 68:3545-3549, 2002). An appropriate combination of a candidate host microbe and the codon-optimized synthetic gene may contribute significantly to establishing a mycotoxin detoxification system for food and feed.

Trichothecene 3-O-acetyltransferase (encoded by Tri101) inactivates the virulence factor of the cereal pathogen Fusarium graminearum. Zearalenone hydrolase (encoded by zhd101) detoxifies the oestrogenic mycotoxin produced by the same pathogen. These genes were introduced into a model monocotyledon rice plant to evaluate their usefulness for decontamination of mycotoxins. The strong and constitutive rice Act1 promoter did not cause accumulation of TRI101 protein in transgenic rice plants. In contrast, the same promoter was suitable for transgenic production of ZHD101 protein; so far, five promising T0 plants have been generated. Low transgenic expression of Tri101 was suggested to be increased by addition of an omega enhancer sequence upstream of the start codon.

A bacterium, strain 314B, able to assimilate ( S)-5-oxo-2-tetrahydrofurancarboxylic acid was isolated from soil and identified as Erwinia cypripedii. A lactonase hydrolyzing ( S)-5-oxo-2-tetrahydrofurancarboxylic acid to l-alpha-hydroxyglutaric acid was purified 63-fold with 2% recovery from crude extracts of this bacterium to homogeneity as judged by SDS-PAGE. The molecular masses estimated by SDS-PAGE and gel filtration were 41 kDa and 79 kDa, respectively. The maximum activity was observed at pH 6.5-7.5 and 35-45 degrees C. The enzyme showed lower activity toward dl-2-oxotetrahydrofuran-4,5-dicarboxylic acid, but did not act on ( R)-5-oxo-2-tetrahydrofurancarboxylic acid and other natural and synthetic lactones tested.