Search Results (21)

An acidic shift in the pH profile of Clonostachys rosea zearalenone hydrolase (ZHD), the most effective and well-studied zearalenone-specific lactone hydrolase, is required to extend the range of applications for the enzyme as a decontamination agent in the feed and food production industries. Amino acid substitutions were engineered in the active center of the enzyme to decrease the pKa values of the catalytic residues E126 and H242. The T216K substitution provided a shift in the pH optimum by one unit to the acidic region, accompanied by a notable expansion in the pH profile under acidic conditions. The engineered enzyme demonstrated enhanced activity within the pH range of 3–5 and improved the activity within the pH ranging from 6 to 10. The D31N and D31A substitutions also resulted in a two-unit shift in the pH optimum towards acidic conditions, although this was accompanied by a significant reduction in the enzyme activity. The D31S substitution resulted in a shift in the pH profile towards the alkaline region. The alterations in the enzyme properties observed following the T216K substitution were consistent with the conditions required for the ZHD application as decontamination enzymes at acidic pH values (from 3.0 to 6.0). Full article

Zearalenone and radicicol are resorcylic acid lactones produced by numerous plant pathogenic fungi. Zearalenone is a non-steroidal estrogen mimic that can cause serious reproductive issues in livestock that consume contaminated feed. Radicicol is a potent inhibitor of the molecular chaperone Hsp90, which, in plants, has an important role in coordinating the host’s immune response during infection. Here, we describe the identification and characterization of a soil-borne strain of the Gram-positive bacterium Aeromicrobium sp. capable of hydrolyzing the macrolide ring of resorcylic acid lactones, including zearalenone and radicicol. Proteomic analysis of biochemically enriched fractions from the isolated and cultured bacterium identified an α/β-hydrolase responsible for this activity. A recombinantly expressed and purified form of the hydrolase (termed RALH) was active against both zearalenone and radicicol. Interpretation of high-resolution mass spectrometry and NMR data confirmed the structures of the enzymatic products as the previously reported non-toxic metabolite hydrolyzed zearalenone and hydrolyzed radicicol. Hydrolyzed radicicol was demonstrated to no longer inhibit the ATPase activity of the Saccharomyces cerevisiae Hsp90 homolog in vitro. Enzymatic degradation of resorcylic acid lactones will enable insight into their biological functions. Full article

Zearalenone (ZEN) is a toxic secondary metabolite produced by the Fusarium fungi, which widely contaminates grains, food, and feed, causing health hazards for humans and animals. Therefore, it is essential to find effective ZEN detoxification methods. Enzymatic degradation of ZEN is believed to be an eco-friendly detoxification strategy, specifically thermostable ZEN degradation enzymes are needed in the food and feed industry. In this study, a novel ZEN lactone hydrolase ZHRnZ from Rosellinia necatrix was discovered using bioinformatic and molecular docking technology. The recombinant ZHRnZ showed the best activity at pH 9.0 and 45 °C with more than 90% degradation for ZEN, α-zearalenol (α-ZOL), β-zearalenol (β-ZOL) and α-zearalanol (α-ZAL) after incubation for 15 min. We obtained 10 mutants with improved thermostability by single point mutation technology. Among them, mutants E122Q and E122R showed the best performance, which retained more than 30% of their initial activity at 50 °C for 2 min, and approximately 10% of their initial activity at 60 °C for 1 min. The enzymatic kinetic study showed that the catalytic efficiency of E122R was 1.3 times higher than that of the wild-type (WT). Comprehensive consideration suggests that mutant E122R is a promising hydrolase to detoxify ZEN in food and feed. Full article

Since the growing number of fungi resistant to the fungicides used is becoming a serious threat to human health, animals, and crops, there is a need to find other effective approaches in the eco-friendly suppression of fungal growth. One of the main mechanisms of the development of resistance in fungi, as well as in bacteria, to antimicrobial agents is quorum sensing (QS), in which various lactone-containing compounds participate as signaling molecules. This work aimed to study the effectiveness of action of enzymes exhibiting lactonase activity against fungal signaling molecules. For this, the molecular docking method was used to estimate the interactions between these enzymes and different lactone-containing QS molecules of fungi. The catalytic characteristics of enzymes such as lactonase AiiA, metallo-β-lactamase NDM-1, and organophosphate hydrolase His₆-OPH, selected for wet experiments based on the results of computational modeling, were investigated. QS lactone-containing molecules (butyrolactone I and γ-heptalactone) were involved in the experiments as substrates. Further, the antifungal activity of the enzymes was evaluated against various fungal and yeast cells using bioluminescent ATP-metry. The efficient hydrolysis of γ-heptalactone by all three enzymes and butyrolactone I by His₆-OPH was demonstrated for the first time. The high antifungal efficacy of action of AiiA and NDM-1 against most of the tested fungal cells was revealed. Full article

Sensory attributes strongly influence consumers’ preferences for products. The inoculation of the Klebsiella variicola H8 strain in a reconstituted tobacco leaf concentrate (RTLC) solution increased neutral aroma-enhancing compound (NAEC) production by 45%, decreased the nicotine level by 25%, decreased the water-soluble total sugar content by ~36%, and improved the sensory quality by 5.71%. The production of NAECs such as dihydrokiwi lactone (DHKL: 192.86%), 1,2,3,4-tetrahydro-1,1,6-trimethylnaphthalene (THTMN: 177.77%), 2,4-di-tert-butylphenol (DTBP: 25%), 4-oxoisofolkone (OIFK: 116.66%,) 1,9-heptadecadiene-4,6-diyn-3-ol (HDD: 116.67%), β-damastrone (BDS: 116.67), and megastigmatrienone A (MSTA: 116.67%) was increased. A metagenomics analysis of the microbial community in the fermented RTLC (FRTLC) was performed to elucidate the mechanism by which NAECs were produced. As a result, 24 groups of functional genes were identified, and among them, five families of carbohydrate-active enzymes, (i) glycoside hydrolase (GH), (ii) glycosyltransferase (GT), (iii) polysaccharide lyase (PL), (iv) carbohydrate esterase (CE), and (v) auxiliary active enzyme (AA), were found to be positively correlated with the production of NAECs. However, among the GHs, the GHs annotated from the H8 strain chromosome displayed the highest relative abundance and a positive correlation with the production of NAECs. Specifically, the GH13-14, GH13-20, GH13-38, GH13-25, GH13-10, GH42, and GH28 genes of the H8 strain were relatively more abundant and were key contributors to the production of NAECs. The correlation analyses revealed that the H8 strain plays a leading role among all the microorganisms in FRTLC in the production of NAECs. Our findings support the application of Klebsiella variicola in NAEC production and a reduction in nicotine content in tobacco products. Full article

Zearalenone (ZEN) is considered one of the most serious mycotoxins contaminating grains and their by-products, causing significant economic losses in the feed and food industries. Biodegradation pathways are currently considered the most efficient solution to remove ZEN contamination from foods. However, low degradation rates and vulnerability to environmental impacts limit the application of biodegradation pathways. Therefore, the main research objective of this article was to screen strains that can efficiently degrade ZEN and survive under harsh conditions. This study successfully isolated a new strain L9 which can efficiently degrade ZEN from 108 food ingredients. The results of sequence alignment showed that L9 is Bacillus velezensis. Meanwhile, we found that the L9 degradation rate reached 91.14% at 24 h and confirmed that the primary degradation mechanism of this strain is biodegradation. The strain exhibits resistance to high temperature, acid, and 0.3% bile salts. The results of whole-genome sequencing analysis showed that, it is possible that the strain encodes the key enzyme, such as chitinase, carboxylesterases, and lactone hydrolase, that work together to degrade ZEN. In addition, 227 unique genes in this strain are primarily involved in its replication, recombination, repair, and protective mechanisms. In summary, we successfully excavated a ZEN-degrading, genetically distinct strain of Bacillus velezensis that provides a solid foundation for the detoxification of feed and food contamination in the natural environment. Full article

Fermented milk products (FMPs) contain probiotics that are live bacteria considered to be beneficial to human health due to the production of various bioactive molecules. In this study, nine artisanal FMPs (kefir, ayran, khurunga, shubat, two cottage cheeses, bryndza, khuruud and suluguni-like cheese) from different regions of Russia were characterized using metagenomics. A metagenomic sequencing of ayran, khurunga, shubat, khuruud and suluguni-like cheese was performed for the first time. The taxonomic profiling of metagenomic reads revealed that Lactococcus species, such as Lc. lactis and Lc. cremoris prevailed in khuruud, bryndza, one sample of cottage cheese and khurunga. The latter one together with suluguni-like cheese microbiome was dominated by bacteria, affiliated to Lactobacillus helveticus (32–35%). In addition, a high proportion of sequences belonging to the genera Lactobacillus, Lactococcus and Streptococcus but not classified at the species level were found in the suluguni-like cheese. Lactobacillus delbrueckii, as well as Streptococcus thermophilus constituted the majority in another cottage cheese, kefir and ayran metagenomes. The microbiome of shubat, produced from camel’s milk, was significantly distinctive, and Lentilactobacillus kefiri, Lactobacillus kefiranofaciens and Bifidobacterium mongoliense represented the dominant components (42, 7.4 and 5.6%, respectively). In total, 78 metagenome-assembled genomes with a completeness ≥ 50.2% and a contamination ≤ 8.5% were recovered: 61 genomes were assigned to the Enterococcaceae, Lactobacillaceae and Streptococcaceae families (the Lactobacillales order within Firmicutes), 4 to Bifidobacteriaceae (the Actinobacteriota phylum) and 2 to Acetobacteraceae (the Proteobacteria phylum). A metagenomic analysis revealed numerous genes, from 161 to 1301 in different products, encoding glycoside hydrolases and glycosyltransferases predicted to participate in lactose, alpha-glucans and peptidoglycan hydrolysis as well as exopolysaccharides synthesis. A large number of secondary metabolite biosynthetic gene clusters, such as lanthipeptides, unclassified bacteriocins, nonribosomal peptides and polyketide synthases were also detected. Finally, the genes involved in the synthesis of bioactive compounds like β-lactones, terpenes and furans, nontypical for fermented milk products, were also found. The metagenomes of kefir, ayran and shubat was shown to contain either no or a very low count of antibiotic resistance genes. Altogether, our results show that traditional indigenous fermented products are a promising source of novel probiotic bacteria with beneficial properties for medical and food industries. Full article

d-pantolactone is an intermediate in the synthesis of d-pantothenic acid, which is known as vitamin B₅. The commercial synthesis of d-pantolactone is carried out through the selective resolution of dl-pantolactone catalyzed by lactone hydrolase. In contrast to a kinetic resolution approach, the deracemization of dl-pantolactone is a simpler, greener, and more sustainable way to obtain d-pantolactone with high optical purity. Herein, an efficient three-enzyme cascade was developed for the deracemization of dl-pantolactone, using l-pantolactone dehydrogenase from Amycolatopsis methanolica (AmeLPLDH), conjugated polyketone reductase from Zygosaccharomyces parabailii (ZpaCPR), and glucose dehydrogenase from Bacillus subtilis (BsGDH). The AmeLPLDH was used to catalyze the dehydrogenated l-pantolactone into ketopantolactone; the ZpaCPR was used to further catalyze the ketopantolactone into d-pantolactone; and glucose dehydrogenase together with glucose fulfilled the function of coenzyme regeneration. All three enzymes were co-expressed in E. coli strain BL21(DE3), which served as the whole-cell biocatalyst. Under optimized conditions, 36 h deracemization of 1.25 M dl-pantolactone d-pantolactone led to an e.e._p value of 98.6%, corresponding to productivity of 107.7 g/(l·d). Full article

Combined use of various antimicrobial peptides (AMPs) with enzymes that hydrolyze the signaling molecules of the resistance mechanism of various microorganisms, quorum sensing (QS), to obtain effective antimicrobials is one of the leading approaches in solving the antimicrobial resistance problem. Our study investigates the lactoferrin-derived AMPs, lactoferricin (Lfcin), lactoferampin and Lf(1-11), as potential partners for combination with enzymes hydrolyzing lactone-containing QS molecules, the hexahistidine-containing organophosphorus hydrolase (His₆-OPH) and penicillin acylase, to obtain effective antimicrobial agents with a scope of practical application. The possibility of the effective combination of selected AMPs and enzymes was first investigated in silico using molecular docking method. Based on the computationally obtained results, His₆-OPH/Lfcin combination was selected as the most suitable for further research. The study of physical–chemical characteristics of His₆-OPH/Lfcin combination revealed the stabilization of enzymatic activity. A notable increase in the catalytic efficiency of action of His₆-OPH in combination with Lfcin in the hydrolysis of paraoxon, N-(3-oxo-dodecanoyl)-homoserine lactone and zearalenone used as substrates was established. Antimicrobial efficiency of His₆-OPH/Lfcin combination was determined against various microorganisms (bacteria and yeasts) and its improvement was observed as compared to AMP without enzyme. Thus, our findings demonstrate that His₆-OPH/Lfcin combination is a promising antimicrobial agent for practical application. Full article

Organophosphorus hydrolase, containing a genetically introduced hexahistidine sequence (His₆-OPH), attracts the attention of researchers by its promiscuous activity in hydrolytic reactions with various substrates, such as organophosphorus pesticides and chemical warfare agents, mycotoxins, and N-acyl homoserine lactones. The application of various carrier materials (metal-organic frameworks, polypeptides, bacterial cellulose, polyhydroxybutyrate, succinylated gelatin, etc.) for the immobilization and stabilization of His₆-OPH by various methods, enables creation of biocatalysts with various properties and potential uses, in particular, as antidotes, recognition elements of biosensors, in fibers with chemical and biological protection, dressings with antimicrobial properties, highly porous sorbents for the degradation of toxicants, including in flow systems, etc. The use of computer modeling methods in the development of immobilized His₆-OPH samples provides in silico prediction of emerging interactions between the enzyme and immobilizing polymer, which may have negative effects on the catalytic properties of the enzyme, and selection of the best options for experiments in vitro and in vivo. This review is aimed at analysis of known developments with immobilized His₆-OPH, which allows to recognize existing recent trends in this field of research, as well as to identify the reasons limiting the use of a number of polymer molecules for the immobilization of this enzyme. Full article

The kinetics and thermodynamics of the enzymatic degradation of zearalenone (ZEN) in degummed corn oil were investigated by analyzing the impacts of temperature, pH, ZEN hydrolase dosage and ZEN concentration on the initial reaction rate. The kinetic study found that the maximum reaction rate was 0.97 μmol × kg⁻¹ min⁻¹, the Michaelis constant (Km) was 11,476 μmol × kg⁻¹ and the Michaelis equation was V = 0.97[S]/(11,476 + [S]). The thermodynamic study showed that the activation energy (Ea) was 70.37 kJ·mol⁻¹, the activation enthalpy change of the reaction (ΔH) > 0, the free energy of activation (ΔG) > 0 and the activation entropy change (ΔS) < 0, indicating the reaction could not be spontaneous. The reaction mechanism of ZEN was studied by a hybrid quadrupole orbitrap mass spectrometer. It was found that ZEN first generated the intermediate G/L/D/W-ZEN+H₂O, followed by generating the intermediate W-ZEN-H₂O under the action of a degrading enzyme. Then, the lactone bond was opened to produce C₁₈H₂₄O₆, and finally the decarboxylation product C₁₇H₂₄O₄ formed automatically. Full article

Mycotoxins are dangerous compounds and find multiple routes to enter living bodies of humans and animals. To solve the issue and degrade the toxicants, (bio)catalytic processes look very promising. Hexahistidine-tagged organophosphorus hydrolase (His₆-OPH) is a well-studied catalyst for degradation of organophosphorus neurotoxins and lactone-containing quorum-sensing signal molecules. Moreover, the catalytic characteristics in hydrolysis of several mycotoxins (patulin, deoxynivalenol, zearalenone, and sterigmatocystin) were studied in this investigation. The best Michaelis constant and catalytic constant were estimated in the case of sterigmatocystin and patulin, respectively. A possible combination of His₆-OPH with inorganic sorbents treated by low-temperature plasma was investigated. Further, enzyme–polyelectrolyte complexes of poly(glutamic acid) with His₆-OPH and another enzymatic mycotoxin degrader (thermolysin) were successfully used to modify fiber materials. These catalytically active prototypes of protective materials appear to be useful for preventing surface contact and exposure to mycotoxins and other chemicals that are substrates for the enzymes used. Full article

The effect of Bacitracin as an antibiotic acting against Gram-positive bacterial cells was evaluated in combination with hexahistidine-containing organophosphate hydrolase (His₆-OPH), possessing lactonase activity against various N-acylhomoserine lactones produced by most Gram-negative bacteria as quorum-sensing molecules. The molecular docking technique was used to obtain in silico confirmation of possible interactions between molecules of His₆-OPH and Bacitracin as well as the absence of a significant influence of such interactions on the enzymatic catalysis. The in vitro experiments showed a sufficient catalytic efficiency of action of the His₆-OPH/Bacitracin combination as compared to the native enzyme. The notable improvement (up to 3.3 times) of antibacterial efficiency of Bacitracin was revealed in relation to Gram-negative bacteria when it was used in combination with His₆-OPH. For the first time, the action of the Bacitracin with and without His₆-OPH was shown to be effective against various yeast strains, and the presence of the enzyme increased the antibiotic effect up to 8.5 times. To estimate the role of the enzyme in the success of His₆-OPH/Bacitracin with yeast, in silico experiments (molecular docking) with various fungous lactone-containing molecules were undertaken, and the opportunity of their enzymatic hydrolysis by His₆-OPH was revealed in the presence and absence of Bacitracin. Full article

N-Acylethanolamine acid amidase (NAAA) is an N-terminal cysteine hydrolase that preferentially catalyzes the hydrolysis of endogenous lipid mediators such as palmitoylethanolamide, which has been shown to exhibit neuroprotective and antinociceptive properties by engaging peroxisome proliferator-activated receptor-α. A few potent NAAA inhibitors have been developed, including α-acylamino-β-lactone derivatives, which are very strong and effective, but they have limited chemical and plasmatic stability, compromising their use as systemic agents. In the present study, as an example of a molecule belonging to the chemical class of N-(2-oxo-3-oxetanyl)amide NAAA inhibitors, URB866 was entrapped in poly(lactic-co-glycolic acid) nanoparticles in order to increase its physical stability. The data show a monomodal pattern and a significant time- and temperature-dependent stability of the molecule-loaded nanoparticles, which also demonstrated a greater ability to effectively retain the compound. The nanoparticles improved the photostability of URB866 with respect to that of the free molecule and displayed a better antioxidant profile on various cell lines at the molecule concentration of 25 μM. Overall, these results prove that the use of polymeric nanoparticles could be a useful strategy for overcoming the instability of α-acylamino-β-lactone NAAA inhibitors, allowing the maintenance of their characteristics and activity for a longer time. Full article

To obtain fiber materials with pronounced chemical-biological protection, metal (Zn or Ta) nanoparticles were jointly applied with polyelectrolyte complexes of enzymes and polypeptides being their stabilizers. Computer modeling revealed the preferences between certain polyelectrolyte partners for N-acyl-homoserine lactone acylase and hexahistidine-tagged organophosphorus hydrolase (His₆-OPH) possessing the quorum quenching (QQ) behavior with bacterial cells. The combinations of metal nanoparticles and enzymes appeared to function better as compared to the combinations of the same QQ-enzymes with antibiotics (polymyxins), making it possible to decrease the applied quantities by orders of magnitude while giving the same effect. The elimination of Gram-positive and Gram-negative bacterial cells from doubly modified fiber materials notably increased (up to 2.9-fold), whereas His₆-OPH retained its hydrolytic activity in reaction with organophosphorus compounds (up to 74% of initially applied activity). Materials with the certain enzyme and Zn nanoparticles were more efficient against Bacillus subtilis cells (up to 2.1-fold), and Ta nanoparticles acted preferentially against Escherichia coli (up to 1.5-fold). Some materials were proved to be more suitable for combined modification by metal nanoparticles and His₆-OPH complexes as antimicrobial protectants. Full article