Search Results (130)

This study aimed to evaluate solid-state fermentation (SSF) as a sustainable approach for the simultaneous detoxification of olive pomace (OP) and the production of industrially relevant enzymes. OP, a semisolid byproduct of olive oil extraction, is rich in lignocellulose and phenolic compounds, which limit its direct reuse due to phytotoxicity. A native strain of Aspergillus sp., isolated from OP, was employed as the biological agent, while grape pomace (GP) was added as a co-substrate to enhance substrate structure. Fermentations were conducted at two scales, Petri dishes (20 g) and a fixed-bed bioreactor (FBR, 2 kg), under controlled conditions (25 °C, 7 days). Key parameters monitored included dry and wet weight loss, pH, color, phenolic content, and enzymatic activity. Significant reductions in color and polyphenol content were achieved, reaching 68% in Petri dishes and 88.1% in the FBR, respectively. In the FBR, simultaneous monitoring of dry and wet weight loss enabled the estimation of fungal biotransformation, revealing a hysteresis phenomenon not previously reported in SSF studies. Enzymes such as xylanase, endopolygalacturonase, cellulase, and tannase exhibited peak activities between 150 and 180 h, with maximum values of 424.6 U·g⁻¹, 153.6 U·g⁻¹, 67.43 U·g⁻¹, and 6.72 U·g⁻¹, respectively. The experimental data for weight loss, enzyme production, and phenolic reduction were accurately described by logistic and first-order models. These findings demonstrate the high metabolic efficiency of the fungal isolate under SSF conditions and support the feasibility of scaling up this process. The proposed strategy offers a low-cost and sustainable solution for OP valorization, aligning with circular economy principles by transforming agro-industrial residues into valuable bioproducts. Full article

Alperujo, a solid by-product from the two-phase olive oil extraction process, poses significant environmental challenges due to its high organic load, phytotoxicity, and phenolic content. At the same time, it represents a promising feedstock for recovering value-added compounds such as phenols and volatile fatty acids (VFAs). When used as a substrate for white rot fungi (WRF), it also produces ligninolytic enzymes. This study explores the use of two native WRF, Anthracophyllum discolor and Stereum hirsutum, for the biotransformation of alperujo under solid-state fermentation conditions, with and without supplementation of copper and manganese, two cofactors known to enhance fungal enzymatic activity. S. hirsutum stood out for its ability to release high concentrations of phenolic compounds (up to 6001 ± 236 mg gallic acid eq L⁻¹) and VFAs (up to 1627 ± 325 mg L⁻¹) into the aqueous extract, particularly with metal supplementation. In contrast, A. discolor was more effective in degrading phenolic compounds within the solid matrix, achieving a 41% reduction over a 30-day period. However, its ability to accumulate phenolics and VFAs in the extract was limited. Both WRF exhibited increased enzymatic activities (particularly Laccase and Manganese Peroxidase) with the addition of Cu-Mn, highlighting the potential of the aqueous extract as a natural source of biocatalysts. Phytotoxicity assays using Solanum lycopersicum seeds confirmed a partial detoxification of the treated alperujo. However, none of the fungi could entirely eliminate inhibitory effects on their own, suggesting the need for complementary stabilization steps before agricultural reuse. Overall, the results indicate that S. hirsutum, especially when combined with metal supplementation, is better suited for valorizing alperujo through the recovery of bioactive compounds. Meanwhile, A. discolor may be more suitable for detoxifying the solid phase strategies. These findings support the integration of fungal pretreatment into biorefinery schemes that valorize agroindustrial residues while mitigating environmental issues. Full article

Glutathione S-transferases (GSTs) are phase II detoxification enzymes that display several enzymatic activities, including transferase, peroxidase, reductase, and isomerase functions, as well as non-enzymatic roles (e.g., serving as binding proteins). Their complex functionality lies in the biotransformation of xenobiotics (e.g., pesticides, drugs) and certain endogenous compounds, primarily metabolites produced by phase I detoxification enzymes. Several plant-derived compounds have been shown to modulate the activity and expression levels of these enzymes. Phytochemical activators of GSTs are potentially beneficial for detoxification in cases of exposure to various toxic compounds, whereas inhibitors of GSTs could have positive effects as adjuvant treatments for cancers that express high levels of GSTs associated with drug resistance. Full article

The aim of this study was to determine whether a low dose of zearalenone (ZEN) affects the mRNA expression of the CYP1A1 (P450 cytochrome) and GSTπ1 (glutathione S-transferase) genes in the large intestine of pre-pubertal gilts. Materials: Control (C) group gilts (n = 18) received a placebo. Experimental (E) group gilts (n = 18) were orally administered 40 μg ZEN/kg body weight (BW) each day before morning feeding for 42 days. Three animals from each group were sacrificed each week of the study. Tissue samples were collected from the medial parts of the ascending colon and the descending colon on six dates. Results: Zearalenone concentrations were multiple times higher in the last three weeks of exposure, and ZEN metabolites were not detected. In phase I, CYP1A1 mRNA expression in the ascending colon was suppressed in the final three weeks of exposure, which substantially increased the ZEN concentration in the descending colon. In phase II, ZEN levels were high in the descending colon due to CYP1A1 suppression in the ascending colon. Consequently, the phase II detoxification processes could not take place due to the absence of a substrate. Conclusion: This study demonstrated that low-dose ZEN mycotoxicosis disrupts the expression of the CYP1A1 and GSTπ1 genes, which co-participate in the enzymatic biotransformation of ZEN in both examined sections of the large intestine. The above could have contributed to increased ZEN accumulation in the mucosa of the descending colon in the last three weeks of exposure. Full article

Background: In order to address complex scenarios in anti-doping science, especially in cases where an unintentional exposure of athletes to prohibited substances and a corresponding contamination of doping control samples at the collection event are argued, an understanding of tissue-specific drug metabolism is essential. Hence, in this study, the metabolic capacity of the seminal vesicle using in vitro assays was investigated. Methods: The aim was to assess whether selected doping-relevant substances—stanozolol, LGD-4033, GW1516, trimetazidine, and anastrozole—are metabolised in seminal vesicle cellular fractions (SV-S9) and how that metabolism compares to biotransformations induced by human liver S9 fractions (HL-S9). Liquid chromatography coupled to high-resolution/accurate mass spectrometry (LC HRAM MS) enabled the sensitive detection and identification of metabolites, revealing a limited metabolic activity of SV-S9. Results: For LGD-4033, GW1516, and trimetazidine, minor metabolic transformations were observed, whereas no metabolites of stanozolol or anastrozole were detected. Gene expression analysis using digital polymerase chain reaction (dPCR) confirmed transcripts of CYP2D6, CYP2E1, and CYP2C9 in SV-S9, though no enzymatic activity was detected. Gene expression and enzymatic activity in CYP3A4 and CYP1A2—major hepatic enzymes—were absent in SV-S9. Conclusions: Overall, these pilot study results suggest that the seminal vesicle has only a low capacity for xenobiotic metabolism, which translates into a limited role in the biotransformation of drugs and, hence, the metabolic pattern. Full article

Background/Objectives: Rhodococcus rhodochrous IEGM 107 cells exhibit pronounced catalytic activity toward mono- and diterpenoids. However, the genetics and enzymatic foundations underlying this activity remain poorly understood. Methods: Using new-generation sequencing, the R. rhodochrous IEGM 107 whole genome was sequenced. Bioinformatic analysis and PCR were employed to identify and characterize genes, with a focus on cytochromes P450 (CYP450s). Results: The catalytic potential of R rhodochrous IEGM 107 was revealed. Its CYP450 genes were detected and analyzed, providing information on the enzymatic base of the strain related to the biotransformation of terpenoids. Conclusions: These findings enhance the understanding of the molecular and genetic basis for terpenoid transformations in R. rhodochrous actinomycetes. The results provide a foundation for future studies on gene expression and enzyme characterization aimed at developing efficient and selective biocatalysts for mono- and diterpenoid transformations. Full article

Phenolic acids, as widely distributed secondary metabolites in plants, possess significant biological activities, such as antioxidant and anti-inflammatory effects. However, their practical applications are limited by low absorption rates and poor bioavailability. Biotransformation technology, with its advantages of strong substrate specificity and mild reaction conditions, has become an effective strategy for the directional modification of phenolic acid molecular structures and the preparation of high-value-added derivatives. Among the various methodologies, enzymatic methods stand out due to their high selectivity and specificity, establishing them as a key approach for phenolic acid biotransformation. The research indicates that coordinated multi-pathway approaches, including decarboxylation, reduction, and hydrolysis, can effectively enhance the efficiency of phenolic acid biotransformation. This review systematically examines the structure and mechanism of action of the key enzymes involved in the phenolic acid biotransformation process. It also proposes innovative pathways and future development directions for existing technologies. Furthermore, it provides an in-depth analysis of the specific application potential of these key enzymes within the food sector. The objective of this review is to furnish a theoretical foundation and technical support for the efficient application of enzymatic methods in phenolic acid biotransformation, thereby accelerating their practical implementation. Full article

The “11th International Congress on Biocatalysis (biocat2024)” was part of a biennial series that unites the fields of biology and chemistry, attracting researchers from the life sciences, engineering, and computer science. This international forum provides an opportunity for scientists worldwide to connect, seek collaboration for future projects, and gain insights into contemporary topics and innovative techniques. Biocat covers a range of compelling subjects and recent advancements in biocatalysis, including enzyme discovery, evolution, and applications. This congress focused on six key topics: AI and computational methods, structure–function analysis and enzyme engineering, enzymatic and whole-cell biotransformations, reaction cascades (electro-, chemo-, and photoenzymatic synergies), bioprocess engineering and the design of smart reactors, and facing climate change through sustainability and a circular bioeconomy. In 2024, we welcomed 344 expert delegates alongside 21 internal attendees, including 154 women and 1 non-binary participant, bringing the total number of participants to an impressive 365. Established researchers and emerging scientists from academia and industry delivered a total of 119 presentations, comprising 59 standard lectures, 60 lightning talks, and 195 posters. Six industry exhibitors showcased their latest products and services, providing an excellent opportunity to strengthen the connection between science and industry. Furthermore, the biocat award, recognized as one of the most prestigious honors in biotechnology, was presented for the eleventh time in the categories of “Science in Academia”, “Lifetime Achievement,” and “Industry”. Full article

The relationships between mycotoxins content in food commodities or feedstuffs and the foodborne diseases is well known. So far, the available data mainly include chemical methods of mycotoxins decontamination for agricultural commodities or raw materials, including mycotoxin binders. Therefore, the possible use of some natural and cost-effective supplements such as herbs, fungi, microorganisms, or plants with powerful and safe protection against mycotoxin-induced health ailments is the main subject of this review paper. Various antagonistic microorganisms or yeast with fungicidal properties, as well as some herbs or plants that suppress fungal development and the subsequent production of target mycotoxins and/or have protective effect against mycotoxins, are deeply studied in the literature, and practical suggestions are given in this regard. The protection by degradation, biotransformation, or binding of mycotoxins by using natural additives such as herbs or plants to feedstuffs or foods has also been thoroughly investigated and analyzed as a possible approach for ameliorating the target adverse effects of mycotoxins. Possible beneficial dietary changes have also been studied to potentially alleviate mycotoxin toxicity. Practical advice are provided for possible application of the same natural supplements in real-life practice for combating mycotoxin-induced health ailments. Natural feed supplements and bioactive compounds appeared to be safe emerging approaches to preventing health ailments caused by mycotoxins. However, the available data mainly address some in vitro studies, and more in vivo experiments are necessary for introducing such approaches in the real-life practice or industry. Generally, target herbal supplements, antioxidants, or polyenzyme complements could be used as powerful protectors in addition to natural mycotoxin binders. Bioactive agents and enzymatic degradation are reported to be very successful in regard to PAT and OTA, whereas antagonistic microorganisms/fungi/yeasts have a successful application against AFs and PAT-producing fungi. Full article

Alfalfa (Medicago sativa L.) saponins (AS), primarily pentacyclic triterpenoids, may reduce methane emissions from goats (Capra hircus L.). This study evaluated the methane-suppressing potential of Aspergillus niger β-glucosidase-modified AS using in vitro rumen fermentation (0.10 mg/mL inoculum, 24 h incubation, gas chromatography detection). Among the 21 alfalfa cultivars, Pegasis (fall dormancy 9) exhibited the highest antioxidant efficacy (half maximum effective concentration 2.13 mg/mL) and the lowest ferric-reducing activity (0.32 μM Fe²⁺/g) (p < 0.05). Fresh/silage AS reduced methane proportions to 4.50–5.21% of total gas, while enzymatic biotransformation further decreased it to 3.34–3.48% (p < 0.05). Methanogen abundance declined by 20.10–44.93%, and general anaerobic fungi declined by 34.22–44.66% compared to untreated AS (p < 0.05). Metabolomics linked methane suppression to six pathways, including zeatin biosynthesis (via nucleotide metabolites accumulation) and prolactin signaling pathway (via bioactive molecules downregulation), suggesting impaired methanogen energy metabolism and hydrogen flux redirection as mechanisms. Enzymatic AS also enhanced volatile fatty acid production, indicating improved fiber digestion. These in vitro findings demonstrate that enzyme-treated AS modulates rumen fermentation through dual methane mitigation and nutrient utilization enhancement, offering a sustainable feed additive strategy for livestock. Full article

In the field of biotechnology, natural compounds isolated from medicinal plants are highly valued; however, their discovery, purification, biofunctional characterization, and biochemical validation have historically involved time-consuming and laborious processes. Two innovative approaches have emerged to more efficiently discover new bioactive substances: the predicted data mining approach (PDMA) and biotransformation-guided purification (BGP). The PDMA is a computational method that predicts biotransformation potential, identifying potential substrates for specific enzymes from numerous candidate compounds to generate new compounds. BGP combines enzymatic biotransformation with traditional purification techniques to directly identify and isolate biotransformed products from crude extract fractions. This review examines recent research employing BGP or the PDMA for novel compound discovery. This research demonstrates that both approaches effectively allow for the discovery of novel bioactive molecules from natural sources, the enhancement of the bioactivity and solubility of existing compounds, and the development of alternatives to traditional methods. These findings highlight the potential of integrating traditional medicinal knowledge with modern enzymatic and computational tools to advance drug discovery and development. Full article

Estrone (E1) is a female hormone present in large quantities in animal farming, which has, in recent decades, resulted in increasing water and soil pollution. Research into its behaviour in the environment has been more focused on water pollution than on soil or soil groups. Three agricultural soils from the Czech Republic—cambisol, fluvisol, and chernozem—were analyzed in a pot experiment to determine their influence on estrone transformation, with laccase, and Mn-oxidoreductases enzymes being measured for this purpose. From the initial concentration of 50 μg·kg⁻¹ soil E1 solution, 1.36 μg·kg⁻¹ were measured on average in the soils after 28 days. There was a clear transition in estrone concentration between 24 h and day 3, reflected in all three soils by increased enzymatic activity. Aside from this, the three soils behaved differently. Results showed that fluvisol was the most different to both cambisol and chernozem. It had the highest enzymatic activity, but also the highest estrone levels in soil at 28 days (5.09 μg·kg⁻¹) vs. cambisol (1.36 μg·kg⁻¹) and chernozem (0.94 μg·kg⁻¹). The removal mechanisms were considered a combination of estrone soil sorption and enzymatic activity, with each soil exhibiting an individual combination of the two. In fluvisol, sorption was considered predominant, thoughenzymatic activity was also relevant; cambisol presented an alternation of sorption and biodegradation, with neither deemed the main mechanism; and chernozem exhibited predominantly high enzymatic activity at the end of the experiment, which resulted in the lowest estrone in soil at the end of the experiment. Overall, all three soils presented good estrone degradation potential through their various soil properties. Full article

Glycosylation is a critical enzymatic modification that involves the attachment of sugar moieties to target compounds, considerably influencing their physicochemical and biological characteristics. This review explored the role of two primary enzyme classes—glycosyltransferases (GTs) and glycoside hydrolases (GHs, glycosidases)—in catalyzing the glycosylation of natural products, with a specific focus on Ganoderma triterpenoids. While GTs typically use activated sugar donors, such as uridine diphosphate glucose, certain GHs can leverage more economical sugar sources, such as sucrose and starch, through transglycosylation. This paper also reviewed strategies for producing novel terpenoid glycosides, particularly recently isolated bacterial GTs and GHs capable of glycosylating terpenoids and flavonoids. It summarized the newly synthesized glycosides’ structures and biotransformation mechanisms, enhanced aqueous solubility, and potential applications. The regioselectivity and substrate specificity of GTs and GHs in catalyzing O-glycosylation (glucosylation) at distinct hydroxyl and carboxyl groups were compared. Furthermore, a special case in which the novel glycosylation reactions were mediated by GHs, including the formation of unique glycoside anomers, was included. The advantages and specific capabilities of GT/GH enzymes were evaluated for their potential in biotechnological applications and future research directions. Novel fungal triterpenoid glycosides produced through various glycosidases and sugars is expected to expand their potential applications in the future. Full article

β-glucuronidase is an important hydrolase, which plays an important role in drug metabolism, clinical diagnostics, and biotransformation. This study focuses on the heterologous expression, isolation, purification, and its enzymatic properties of β-glucuronidase CpGUS from Clostridium perfringens, as well as its application in the whole-cell transformation of unconjugated bilirubin from pig bile. A recombinant E. coli BL21(DE3)/pET-28a-CpGUS was constructed for the heterologous expression of CpGUS, with the majority of the expressed enzyme being soluble. Enzymatic analysis showed that CpGUS displayed optimal activity at pH 5.0 and 45 °C, and it rapidly lost activity at pH < 4.5. Metal ions, such as Mg²⁺ and Fe²⁺, enhanced CpGUS catalysis, while Zn²⁺, K⁺, Fe³⁺, Mn²⁺, Cu²⁺, and Na⁺ inhibited it. Notably, Cu²⁺ and Fe³⁺ can significantly inhibit β-glucuronidase, resulting in the complete loss of its activity. The results of the whole-cell transformation experiment show that when E.coli BL21(DE3)/ pET-28a-CpGUS at an OD₆₀₀ of 10 was incubated at pH 5.0, a temperature of 45 °C, and a rotation speed of 200 rpm for 12 h, the hydrolysis rate of the conjugated bilirubin in pig bile reached 81.1%, the yield of unconjugated bilirubin was 76.8%, and the purity of unconjugated bilirubin was 98.2%. The three-dimensional structure of CpGUS was predicted using AlphaFold2 (AlphaFold v2.0, DeepMind Technologise Limited, London, UK), and p-Nitrophenyl-β-D-Glucuronide (pNPG) and conjugated bilirubin were then docked to the CpGUS protein model using SWISSDOCK. The best docked conformations of the CpGUS–pNPG and CpGUS–conjugated bilirubin complex systems were simulated by independent 500 ns molecular dynamics (MD) runs with the RSFF2C force field, and the binding dynamic and catalytic mechanism of each system were obtained. The results indicated that π-π stacking, hydrogen bonding, and hydrophobic interactions between the key residue Tyr472 and the benzene ring of pNPG molecules are crucial for its catalytic process. Similarly, for the binding and catalysis of conjugated bilirubin by CpGUS, the π-π stacking and hydrogen bonding and hydrophobic interactions between the sidechains of residues Phe368 and Tyr472 and the benzene ring of conjugated bilirubin play a synergistic role during its catalytic process. Their total binding free energy (∆G_bind) values were calculated to be as high as −65.05 ± 12.66 and −86.70 ± 17.18 kJ/mol, respectively. These results suggest that CpGUS possesses high binding and catalytic hydrolysis properties for both pNPG and conjugated bilirubin. Full article

The metabolism of drugs and foreign substances in humans typically involves multiple enzymatic steps, particularly in phase-1 biotransformation in the liver, where various cytochrome P450 monooxygenases (CYPs) play crucial roles. This complexity can lead to a wide range of metabolites. Understanding the contributions of individual CYPs and their interactions within these intricate enzyme cascades can be challenging. We recently developed an in vitro biotransformation platform employing various Chinese Hamster Ovarian (CHO) cell clones. These clones express human cytochrome P450 oxidoreductase (CPR), and each is defined by a specific human CYP enzyme expression, thus exhibiting no detectable endogenous CYP enzyme activity (mono-CYP CHO platform). In this study, we investigated whether the mono-CYP CHO platform is a suitable tool for modeling complex drug metabolization reactions in vitro. Tamoxifen (TAM) was selected as a model substance due to its role as a prodrug widely used in breast cancer therapy, where its main active metabolite, endoxifen, arises from a two-step metabolism primarily involving the CYP system. Specifically, the combined activity of CYP3A4 and CYP2D6 is believed to be essential for efficient endoxifen production. However, the physiological metabolization pathway of TAM is more complex and interconnected, and the reasons for TAM’s therapeutic success and variability among patients are not yet fully understood. Analogous to our recently introduced mono-CYP3A4 CHO cells, we generated a CHO cell line expressing human CPR and CYP2D6, including analysis of CYP2D6 expression and specific activity. Comparative studies on the metabolization of TAM were performed with both mono-CYP CHO models individually and in co-culture with intact cells as well as with isolated microsomes. Supernatants were analyzed by HPLC to calculate individual CYP activity for each metabolite. All the picked mono-CYP2D6 clones expressed similar CYP2D6 protein amounts but showed different enzyme activities. Mono-CYP2D6 clone 18 was selected as the most suitable for TAM metabolization based on microsomal activity assays. TAM conversion with mono-CYP2D6 and -3A4 clones, as well as the combination of both, resulted in the formation of the expected main metabolites. Mono-CYP2D6 cells and microsomes produced the highest detected amounts of 4-hydroxytamoxifen and endoxifen, along with N-desmethyltamoxifen and small amounts of N,N-didesmethyltamoxifen. N-desmethyltamoxifen was the only TAM metabolite detected in notable quantities in mono-CYP3A4, while 4-hydroxytamoxifen and endoxifen were present only in trace amounts. In CYP2D6/3A4 co-culture and equal mixtures of both CYP microsomes, all metabolites were detected at concentrations around 50% of those in individual clones, indicating no significant synergistic effects. In conclusion, our mono-CYP CHO model confirmed the essential role of CYP2D6 in synthesizing the active TAM metabolite endoxifen and indicated that CYP2D6 is also involved in producing the by-metabolite N,N-didesmethyltamoxifen. The differences in metabolite spectra between the two mono-CYP models highlight the CYP specificity and sensitivity of our in vitro system. Full article