Zearalenone (ZEN) is a prevalent estrogenic mycotoxin that contaminates cereal-based food and feed and poses a substantial risk to human and animal health. Enzymatic detoxification has emerged as a promising mitigation strategy, and several α/β-hydrolases have been shown to efficiently hydrolyze ZEN (EC 3.1.1.B12). Moreover, indirect screening has identified additional structurally distinct enzymes with potential ZEN-degrading activity. In the present study, we evaluated two such enzymes-a Streptomyces exfoliatus lipase (SeLIP) and a Thermobifida cellulosilytica cutinase (TcCUT)-and compared their properties with two established zearalenone hydrolases from Clonostachys rosea and Rhodococcus erythropolis (CrZHD and ReZHD). The enzymes were characterized for thermal stability, and ZEN-degrading capacity was evaluated by direct enzymatic assay combined with LC-MS/MS. Notably, only CrZHD and ReZHD catalyzed ZEN hydrolysis, resulting in complete substrate conversion. The four enzymes were assayed for general esterase activity using p-nitrophenyl acetate (pNPA). All four enzymes exhibited measurable esterase activity toward pNPA, confirming that SeLIP and TcCUT are catalytically competent despite lacking ZEN hydrolytic activity. Structural comparison and molecular docking analyses suggested that TcCUT and SeLIP bind ZEN weakly, in non-productive conformations, likely due to the absence of a cap domain, explaining their lack of ZEN hydrolytic activity. Our results demonstrate that indirect assays based on general esterase activity cannot predict zearalenone hydrolytic activity, leading to false-positive candidate identification. Collectively, these findings highlight the value of a direct LC-MS/MS workflow for distinguishing true ZEN hydrolases from unrelated esterases, enabling more reliable candidate selection for biotechnological applications.

Salmonella enterica and Escherichia coli–associated enteric disturbances contribute to health and productivity losses in poultry and pose zoonotic concerns. In the context of antimicrobial resistance, phytogenic feed additives may offer antibiotic-sparing strategies by modulating the intestinal microbiota. We evaluated a fenugreek (Trigonella foenum-graecum) and turmeric (Curcuma longa) extract combination for its microbiota-modulating effects in a controlled dual-challenge model. A total of 270 Bábolna Tetra-SL chicks were allocated to six groups: low-, medium-, or high-dose phytobiotic; enrofloxacin; infected control; and non-infected control. Birds were orally challenged on days 3–4 post-hatch with clinical isolates of S. enterica and E. coli (both phenotypically susceptible to enrofloxacin). Cloacal swab samples were collected on days 1, 7, and 42 and profiled by V3-V4 16 S rRNA gene amplicon sequencing. Alpha diversity and beta diversity were assessed in QIIME2 using non-parametric and permutation-based approaches. Alpha diversity increased with age across groups. On day 42, the medium-dose phytobiotic group exhibited the most balanced community profile among treated groups, whereas enrofloxacin was associated with the strongest early community disruption followed by partial recovery by day 42. Beta diversity ordinations and clustering indicated clear time-driven separation, with treatment-associated differences observed within time points and supported by permutation-based multivariate statistics. A fenugreek–turmeric phytobiotic modulated cloacal microbiota structure in chickens under a controlled dual-challenge model. Medium-dose supplementation was associated with the most balanced community configuration at the end of the trial, while enrofloxacin induced marked early perturbation. These findings support further evaluation of phytogenic additives as components of antibiotic-reduction strategies in poultry production.

The interaction between modified forms of deoxynivalenol (DON), such as DON-3-glucoside (DON-3G), and feed enzymes is underestimated. We assessed the interaction between DON and non-starch polysaccharide enzymes (NSPases) on growth performance, and excreta levels of DON and its metabolites (Experiment 1), as well as DON toxicokinetics (Experiment 2). Broiler chickens were fed maize-based diets naturally contaminated with low (LD; 516–792 μg DON/kg diet) or moderate (MD; 2395–3020 μg DON/kg diet) levels of DON. The LD and MD diets were supplemented with or without an NSPase blend. The MD diet impaired growth performance in broiler chickens up to 14 days of age, whereas no differences were observed in older birds. NSPase supplementation decreased body weight gain (BWG) in 28-day-old broiler chickens fed the MD diet and increased excreta levels of DON-3-sulfate (DON-3S). NSPase supplementation also improved the feed conversion ratio (FCR) without increasing BWG. Additionally, NSPase altered the toxicokinetic profile of DON in broiler chickens fed the MD diet, and the time to maximum plasma concentration (Tmax) of DON-3S was influenced by both NSPase supplementation and the DON level in the diet. To our knowledge, these findings provide the first evidence that NSPases influence DON toxicokinetics in poultry.

Introduction:Combating antimicrobial resistance is one of the most pressing public health challenges of our time. The rapid spread of resistant, zoonotic bacterial strains in livestock farming is increasingly raising concerns about the need to reduce antibiotic use. Because of this, there is an urgent need for safe and effective alternatives in animal husbandry. Methods:This study aimed to perform an in vivo the dose-response analysis of fenugreek (Trigonella foenum-graecum), as a plant-based antibiotic alternative feed supplement in Bábolna Tetra-SL chicks (1-42 days old) with a 1:1 sex ratio. A total of 270 chicks were randomly assigned to 18 groups (15 birds per group) and subjected to six different treatment groups in three replicates: fenugreek at 1×, 10×, and 100× doses, an antibiotic-treated group (enrofloxacin), a positive control group (infection only), and a negative control group (no infection or treatment). The infection was induced using mixed Salmonella Enteritidis and Escherichia coli, administered via gavage on days 3 and 4 of life. The birds were monitored for clinical symptoms, body weight, feed intake, and Salmonella shedding through cloacal swab samples. Statistical analyses included mixed-effect logistic regression for mortality, mixed-effect linear models for weight gain, two-way ANOVA for feed efficiency, and random effects continuation ratio models for Salmonella isolation. Results:Significant interactions for Group:Day and Sex:Day in weight gain were identified (p < 0.0001 for both). Additionally, the 1 × dose group showed significantly reduced Salmonella shedding compared to the positive control group on day 33 (p = 0.0031). The low-dose group (1×) demonstrated the most promising results, showing a 63% reduction in Salmonella shedding on day 10 and 31% on day 17. This group exhibited the fewest clinical symptoms, no diarrhea, and the lowest individual and specific feed intake up to day 24. Discussion:The findings of this study suggest that low-dose fenugreek supplementation could be a viable strategy for reducing Salmonella shedding in poultry, potentially contributing to reduced antibiotic use in poultry farming and thus playing a role in the global effort to combat antimicrobial resistance. Future research will involve large-scale industrial trials and next-generation sequencing to evaluate the additive's impact on gut microbiota composition.

Fumonisins are sphingolipid-like mycotoxins that cause serious damage by contaminating food and feed. The tricarballylic acid (TCA) units of fumonisin B-1 (FB1; accounting for 70 % of fumonisin contamination) can be removed by fumonisin B-1 esterase (FE, EC 3.1.1.87) providing a biotechnological FB1 detoxification possibility. Here, we report the regioselective cleavage of the TCA ester at C6 in the first step of FB1 hydrolysis and kinetic characterization for two FEs. The low K-M values (4.76-44.3 mu M) are comparable to concentrations of environmental contaminations, and the high catalytic efficiencies are promising for practical applications. The X-ray structure of one of the FEs enabled the understanding of the FB1 hydrolysis at molecular level and revealed an arginine pocket key for substrate binding, and the catalytic role of the glutamate preceding the catalytic serine. Computations showed that this FE is likely capable of detoxifying any fumonisin indicating its potential applicability in food and feed products.

To compensate the lack of molecular-phylogenetic data on hindgut flagellates in Hungary, galliform birds were monitored in five regions for two years. Samples were collected from 11 turkeys (from 4 flocks) and 9 pheasants (from 3 farm-raised flocks) suspected to have histomonosis. These samples were molecularly and phylogenetically analysed. In nine turkeys, five 18S rRNA genes and two ITS sequence variants of Histomonas meleagridis were identified. These variants were identical between the caecum and liver of the same bird in most cases, but different 18S variants were identified between sampling sites. In one turkey, an unnamed species, here designated as "Dientamoebidae sp. HUN35", was identified. Its 18S rRNA gene sequence was near-identical (99.6-99.3%) to the sequence reported previously; and the ITS-1/5.8S/ITS-2 region confirmed a close relationship with H. meleagridis and Dientamoeba fragilis. In one pheasant, Tetratrichomonas gallinarum was detected. Different 18S rRNA variants had either identical or different ITS sequences, thus optimally, both should be used for molecular epidemiological studies. Our results suggest the unnamed Dientamoebidae sp. has been present in Hungary since its first detection in 2010 and the host range of this species as well as that of T. gallinarum is broader than previously thought.

Necrotic enteritis, caused by Clostridium perfringens (C. perfringens) is a disease present worldwide and causes major economic losses. The re-emergence of the disease, in recent years, is mainly due to the ban of the usage of antibiotics as growth promoters in the EU. The aim of this study was to establish a reliable, robust challenge model. Ross hybrid broilers were divided into randomized groups: a positive and a negative control group, a group receiving antibiotic treatment and three groups fed with assorted feed supplements, all receiving the same basal diet. The birds in the treatment groups were vaccinated twice using a 10-times dose of an Infectious Bursitis live vaccine and the animals were challenged four times with a NetB toxin producing C. perfringens strain. The presence of clinical signs and body weight gain were monitored. At the end of the study necropsy was performed and the gut lesions were scored. During the experiment, clinical signs were absent in the negative control group and in the antibiotic treated group. The other animals displayed diarrhea and feather loss. These symptoms were the most pronounced in the positive control group. The gut lesion scores showed significant differences between the negative and positive control groups, with the former scoring the lowest. Based on these results, the challenge model establishment was successful and in this setup the assessment of the potency of feed additives is also possible.

Toxicity of fumonisin B1 (FB1), the most common fumonisin mycotoxin, can be significantly decreased by enzymatic cleavage of its two tricarballylic acid groups, using fumonisin esterase. An enzyme product should be formulated in a solid form for use as a feed supplement, and have a long shelf-life and high heat stability. Therefore, a fumonisin esterase (further referred to as FE2) was expressed in Pichia pastoris and the enzyme was immobilized with different techniques. Covalent immobilization of FE2 on aminefunctionalized zeolite, crosslinked with glutaraldehyde, resulted in low specific capacity (0.8 mg enzyme/g zeolite), and poor stability. Reversible immobilization of FE2 on different solid carriers resulted in even > 95% immobilization efficiency, and the enzyme could be recovered after resuspension in buffer, without significant loss of activity. This immobilization method resulted in at least 3 years of shelf-life even at 50 °C. The thermal stability of these products significantly increased compared to the enzyme solution; the products retained even nearly 100% of their activity after incubating at 100 °C for 30 minutes. This increased heat stability makes the product compatible with feed processing techniques, such as pelleting.

Industrial enzyme production with the Pichia pastoris expression system requires a well-characterized production strain and a competitively priced fermentation medium to meet the expectations of the industry. The present work shows a workflow that allows the rapid and reliable screening of transformants of single copy insertion of the target production cassette. A constitutive expression system with the glyceraldehyde-3-phosphate dehydrogenase promoter (pGAP) with homology arms for the glycerol kinase 1 (GUT1) was constructed for the targeted integration of the expression plasmid in a KU70 deficient Pichia pastoris and the production of a bacterial fumonisin esterase enzyme (CFE). A robust colony qPCR method was developed for the copy number estimation of the expression cassette. Optimization of the protein production medium and the scale-up ability was aided by design of experiments (DOE) approach resulting in optimized production conditions at a semi-industrial scale. A novel fermentation medium containing 3% inactivated yeast and 2% dextrose in an ammonium-citrate buffer (IYD) was shown to be a promising alternative to YPD media (containing yeast extract, peptone, and dextrose), as similar protein titers could be obtained, while the cost of the medium was reduced 20-fold. In a demonstration-scale 48 h long fed-batch fermentation, the IYD media outperformed the small-scale YPD cultivation by 471.5 ± 22.6%.

The purpose of this study was to determine the effectiveness of a phytobiotic-prebiotic feed additive (PPFA, which contains a combination of chicory and extracts of carob pulp and fenugreek) in the diets of fattening pigs on growth indicators, carcass characteristics, and fecal microbiota. A total of 329 crossbred pigs were randomly divided into two dietary treatments, including a basal diet without additives as the control group and a basal diet supplemented with 1 kg/T PPFA as the trial group. The PPFA supplementation led to a significant increase in the body weight gain and average daily gain of the trial group compared to those of the control group after 70 days of feeding. Through the S-EUROP evaluation system, we also found that the fattening pigs fed PPFA significantly improved their carcass indicators. Furthermore, it was shown that PPFA regulated porcine intestinal microbiota, including promoting the growth of the beneficial commensal bacteria (i.e., Bifidobacterium and Lactobacillus) while inhibiting some potential pathogen bacteria (i.e., Bacteroidaceae and Campylobacteraceae). Our work revealed that the phytobiotic-prebiotic feed additive containing carob pulp, chicory, and fenugreek positively influences the intestinal microbiota, growth performance, and carcass traits in fattening swine.

Restrictions on the use of antimicrobial compounds have led to a surge of interest in alternative solutions, such as natural, plant-based compounds. In our study, we investigated the efficacy of three feed supplements containing different additives, namely, probiotics (Lactobacillus spp., “Test substance A”), turmeric (Curcuma longa L., “Test substance B”), and fenugreek (Trigonella foenum graecum, “Test substance C”). In the experiment, we tested 180 birds of the Bábolna Tetra-SL laying hybrid breed that were infected with Salmonella enteritidis strains. The birds were randomly divided into six groups: three groups treated with the different additives, a negative control group, a positive control group, and an antibiotic-treated group using enrofloxacin. We examined the maturation and the time course of shedding of Salmonella; at the end of rearing, pathological and histopathological examinations were performed. When Salmonella was isolated from the cloacal swab samples, the enrofloxacin-treated group had a high number of animals shedding Salmonella by day 9, which was like the group treated with test material C. The greatest reduction in Salmonella shedding was observed in the groups treated with test materials A and B. In terms of pathological parameters, villus length and crypt depth were significantly better in the group treated with test material C compared to the positive and negative controls, and when comparing the body weight of the tested animals, the group treated with test material B had a significantly larger absorption surface area compared to the positive control group. Overall, the supplement with test material C proved to be the most effective. In the future, it is worthwhile to investigate the combination of the tested active substances for their possible synergistic effects and to perform a dose-response study to select the optimal dosage.

This study compared the minimal inhibition concentrations (MICs) and their effects on the growth kinetics of seven different types of zinc (Zn) compounds and Na2EDTA in the case of three typical commensal beneficial microorganisms (Bacillus subtilis, Lactococcus lactis, and Saccharomyces cerevisiae). The seven Zn compounds included ZnSO4, four Zn–amino acid chelates, and two Zn–EDTA complexes. Both MICs and growth kinetic parameters indicated that different microorganisms show different sensitivities; for example, B. subtilis, L. lactis, and S. cerevisiae were most sensitive to ZnSO4, Na2EDTA, and Zn(NH3)2(Gly)2, respectively. Both ZnEDTA and Zn(NH3)2(Lys)2 improved the growth rate of all beneficial commensal intestinal microorganisms at low concentrations (5–10 mg/L) and showed low toxicity towards all tested strains. At higher concentrations (100–500 mg/L), all compounds decreased the growth rate and increased the lag phase. In conclusion, both growth kinetic parameters and MICs tested effectively measured the inhibitory effects of the test materials; however, growth kinetics provides a more detailed picture of the concentration-dependent effects and those on the mechanisms of microbial growth inhibition.

A wide range of phytobiotic feed additives are available on the market claiming to have beneficial effects on the growth of the host animal and to promote the development of a balanced microflora. The present study investigated the effects of the phytobiotic-prebiotic mixture of curcumin, wheat germ, and chicory on the growth performance and on the intestinal microflora composition of weaning piglets. Post weaning diarrhea causes significant losses for the producers, most commonly it is prevented by feeding high doses of zinc oxide (ZnO). The effect of a phytobiotic-prebiotic feed additive (1 kg T-1) was compared to a positive control (3.1 kg T-1 ZnO) and to a negative control (no feed supplement) in an in vivo animal trial. There was no significant difference in the final body weight and average daily gain of the trial and positive control groups, and both groups showed significantly (P<0.05) better results than the negative control. The feed conversion ratio of the phytobiotic-prebiotic supplemented group was significantly improved (P<0.05) compared to both controls. Both phytobiotic-prebiotic mixture and ZnO were able to significantly reduce (P<0.05) the amount of coliforms after weaning, even though ZnO reduced the amount of coliforms more efficiently than the trial feed additive, it also reduced the amount of potentially beneficial bacteria. Metagenomic data also corroborated the above conclusion. In the trial and positive control groups, the relative abundance of Enterobacteriaceae decreased by 85 and 88% between 3 weeks and 6 weeks of age, while in the negative control group a slight increase occurred. Lactobacillaceae were more abundant in the trial group (29.98%) than in the positive (8.67%) or in the negative (22.45%) control groups at 6 weeks of age. In summary, this study demonstrated that a phytobiotic-prebiotic feed additive may be a real alternative to ZnO for the prevention of post weaning diarrhea and promote the development of a balanced gut system.

Nowadays, protein purification by the aid of affinity tags can be carried out with high speed and efficiency. However, in several cases, affinity tags can significantly alter the key properties of enzymes, especially activity and/or thermostability. This study focused on the purification of the non-tagged phenylalanine ammonia-lyase from Petroselinum crispum (PcPAL), as well as on the purification of the TEV (Tobacco Etch Virus) protease, the molecular scissors used to remove the affinity tag from the recombinantly expressed PcPAL. Removal of the 6xHis-tag led to a 1.5-fold increase in the specific activity of PcPAL, while the absence of the affinity tag did not significantly alter the thermostability of the protein. The purity and oligomerization state of the proteins of interest were also analyzed by size exclusion chromatography, both before and after the removal of the affinity tag, confirming the stability of the tetrameric fold of PcPAL

The enzyme family harboring the post-translationally formed 5-methylene-3,5-dihydro-4H-imidazol-4-one (MIO) catalytic residue comprises both aromatic amino acid ammonia-lyases (ALs) and 2,3-aminomu...

Evolutionarily related hydroxynitrile lyases from rubber tree (HbHNL) and from Arabidopsis thaliana (AtHNL) follow different catalytic mechanisms with opposite enantioselectivity toward mandelonitrile. We hypothesized that the HbHNL-like mechanism evolved from an enzyme with an AtHNL-like mechanism. We created ancestor-like composite active-sites in each scaffold to elucidate how this transition may have occurred. Surprisingly, a composite active site in HbHNL maintained (S)-selectivity, while the identical set of active site residues in AtHNL maintained (R)-selectivity. Composite active-site mutants that are (S)-selective without the Lys236 and Thr11 that are required for the classical (S)-HNL mechanism suggests a new mechanism. Modeling suggested a possibility for this new mechanism that does not exist in modern enzymes. Thus, the last common ancestor of HbHNL and AtHNL may have used an extinct mechanism, not the AtHNL-like mechanism. Multiple mechanisms are possible with the same catalytic residues and residues outside the active site strongly influence mechanism and enantioselectivity.

Aromatic amino acid ammonia-lyases and 2,3-aminomutases contain the post-translationally formed prosthetic 3,5-dihydro-4-methylidene-5H-imidazol-5-one (MIO) group. The so-called MIO-enzymes may be used for the stereoselective synthesis of enantiopure α- or β-amino acids, making these chemical processes more environmentally friendly and more affordable. Even though, a number of structures are available in the PDB for MIO enzymes, the only structure for a eukaryotic phenylalanine ammonia-lyase (PAL) (PDB:1W27) is in catalytically inactive conformation, due to the “loop-out” conformation of catalytically important Tyr-loop.

This study describes the cloning of the gene encoding a novel phenylalanine ammonia-lyase from Kangiella koreensis (KkPAL) into pET19b expression vector.Optimization of protein expression and purification conditions yielded 15 mg pure soluble protein from one liter of E. coli culture.Enzymatic activity measurements of the ammonia elimination reaction from different natural aromatic amino acids proved the protein to be a phenylalanine ammonia-lyase.The isolated protein showed remarkably high, 81.7 °C melting temperature, making it especially suitable for biocatalytic applications.

Aromatic amino acid ammonia-lyases and aromatic amino acid 2,3-aminomutases contain the post-translationally formed prosthetic 3,5-dihydro-4-methylidene-5H-imidazol-5-one (MIO) group. MIO enzymes catalyze the stereoselective synthesis of α- or β-amino acid enantiomers, making these chemical processes environmentally friendly and affordable. Characterization of novel inhibitors enables structural understanding of enzyme mechanism and recognizes promising herbicide candidates as well. The present study found that both enantiomers of the aminophosphonic acid analogue of the natural substrate phenylalanine and a novel derivative bearing a methylidene at the β-position inhibited phenylalanine ammonia-lyases (PAL), representing MIO enzymes. X-ray methods unambiguously determined the absolute configuration of all tested enantiomers during their synthesis. Enzyme kinetic measurements revealed the enantiomer of the methylidene-substituted substrate analogue as being a mirror image relation to the natural l-phenylalanine as the strongest inhibitor. Isothermal titration calorimetry (ITC) confirmed the binding constants and provided a detailed analysis of the thermodynamic driving forces of ligand binding. Molecular docking suggested that binding of the (R)- and (S)-enantiomers is possible by a mirror image packing.