Search Results (164)

Cadmium (Cd) toxicity destroys plant cells and affects plant growth and development. Due to its unique metallic properties, selenium (Se) has been shown to be effective in antioxidants, cellular immunity, and heavy metal detoxification. When Se and Cd are present together in plants, they antagonize. However, the mechanism of action of the two in the rice cell wall remains to be clarified. In this study, we analyzed the mechanism of Cd detoxification by rice (Oryza sativa L.) cellular polysaccharides mediated by Se, using the cell wall as an entry point. Proteomic and transcriptomic analyses revealed that “Glycosyl hydrolases family 17”, “O-methyltransferase”, and “Polygalacturonase” protein pathways were significantly expressed in the cell wall. The most abundant enzymes involved in polysaccharide biosynthesis were found, including bglB, otsB, HK, PFP, ADH1, and ALDH, which resulted in the synthetic pathway of polysaccharide formation in the rice cell wall. Finally, the essential genes/proteins, such as protein Os03g0170500, were identified. The study showed that Se inhibits Cd uptake and transport when Se (1 mg/kg) is low relative to Cd (3 mg/kg), has little inhibitory effect, and even promotes Cd (3 mg/kg) uptake when Se (5 mg/kg) is relatively high. Full article

Fungal cell walls, composed of polysaccharides and proteins, play critical roles in adaptation, cell division, and protection against environmental stress. Their polyglucan components are continuously remodeled by various types of glycosyl hydrolases (GHs) and transferases (GTs). In Saccharomyces cerevisiae and other ascomycetes, enzymes of the Dfg5 subfamily, which belong as GTs to the GH76 family, cleave an α1,4 linkage between glucosamine and mannose to facilitate covalent linkage of GPI-anchored proteins to the cell wall’s polyglucans. In contrast, the functions of other fungal GH76 subfamilies are not understood. We characterized CtGH76 from the sordariomycete Chaetomium thermophilum, a member of the Fungi/Bacteria-mixed GH76 subfamily, revealing conserved structural features and functional divergence within the GH76 family. Notably, our structural characterization by X-ray crystallography combined with glycan fragment screening indicated that CtGH76 can recognize GPI-anchors like members of the Dfg5 subfamily but shows a broader promiscuity toward other glycans with central α1,6-mannobiose motifs due to the presence of an elongated glycan-binding canyon. These findings provide new insights into GH76 enzyme diversity and fungal cell wall maturation. Full article

Acinetobacter baumannii is an opportunistic pathogen and a major cause of nosocomial infections worldwide. This study aimed to isolate and characterize phages with lytic activity against multidrug-resistant A. baumannii strains to enable antibacterial alternatives. Eight phages (AKO8a, PS118, B612, MCR, IDQ7, 89P13, CRL20, and CIM23) were isolated and subjected to genomic, phylogenetic, and functional analyses. Antibacterial activity was assessed in vitro against A. baumannii strain AbAK04 by measuring optical density over 17 h at multiplicities of infection (MOIs) of 0.1, 1, and 10, using a repeated-measures design with time as a crossed factor and MOI as a nested factor. Tukey’s post-hoc test identified significant bacterial growth reductions of 57–72% (p < 0.001). Specifically, phages PS118 and 89P13 reduced growth by 71% at MOI 10; CIM23, B612, and CRL20 achieved 68% reduction at MOI 1; and MCR reduced growth by 64% at MOIs 0.1 and 1. Notably, lytic phage MCR encodes a glycosyl hydrolase family 58 (GH58) enzyme, potentially contributing to its antibacterial activity. Genomic analyses confirmed absence of virulence and antibiotic resistance genes, with all phages classified as novel species within the Kagunavirus genus. These findings support the use of these phages as promising candidates for in vivo evaluation. Full article

Annually, a considerable amount of agricultural waste is produced leading to serious environmental pollution if not managed effectively. A wide range of bio-decomposers, including fungi, bacteria, and actinomycetes may break down the complex agro-residues in an eco-friendly way through secreting many cellulolytic and amylolytic enzymes. The present study aimed at exploring the ability of Frankia to degrade cellulose and starch and identifying the cellulase and α-amylase genes in Frankia genomes for potential agricultural waste degradation. Frankia alni ACN14a and Frankia casuarinae CcI₃ produced clear zones around growing hyphae on carboxymethyl cellulose (CMC) and starch substrates. The hydrolytic index (HI) ranged from 1 to 2.14 reflecting variation in their degradation efficacy. Quantification of CMCase (carboxymethyl cellulase) production in strain ACN14a presented the maximum activity (0.504 U/mL) under 1% CMC after 16 days whereas strain CcI₃ produced a weak activity after 6 days from incubation. Besides, amylase activity in strain ACN14a reached the highest value (3.215 U/mL) after 4 days of growing with 1% starch, while strain CcI₃ had the superior production (3.04 U/mL) after 12 days from 1% starch condition. Data mining and genome blasting led to the identification of multiple genes related to cellulose and starch degradation. Two endoglucanases (celA1, FRAAL4955 and celA2, FRAAL4956), two glycosyl hydrolase family 16 (FRAAL6120 and FRAAL2663), and one glycosyl hydrolase family 16 (Francci3_3843) were predicted in the two genomes. Likewise, the α-amylase genes (FRAAL5900) from Frankia alni ACN14a and (Francci3_3679) from strain CcI₃ were identified. The gene expression of endo-1, 4-beta-glucanase (celA2, FRAAL4956) revealed the maximum increment in its mRNA abundance under 0.25% CMC exposure and showed a 3.3-fold increase. Frankia capability to degrade cellulose and starch represents a critical process in nutrient cycling and environment protection. Full article

β-Galactosidase, a glycoside hydrolase enzyme, also possesses glycosyl transferase activity and can glycosylate various aglycones, including tyrosol, a phenylethanoid with antioxidant and health-promoting effects. This study examines the effect of lactose, tyrosol and deep eutectic solvents (DESs) as co-solvents on the stability and activity of Aspergillus oryzae β-galactosidase during the enzymatic synthesis of tyrosol β-d-galactoside (TG). The enzyme’s thermal stability was assessed using nanoDSF and circular dichroism spectroscopy, while the enzyme’s activity and specificity toward different glycosyl acceptors were investigated using the initial rate method. The effects of tyrosol and DESs on tyrosol galactoside synthesis over a 6 h period were also studied. Lactose and glycerol were found to stabilize the enzyme. Among the DESs tested, those containing betaine showed the highest stabilizing effect. The presence of DESs not only affected the overall enzyme activity but also changed the enzyme specificity, most frequently in favor of lactose hydrolysis. Components of DESs containing alcohol groups (polyols) also acted as transglycosylation acceptors. However, both glycerol and tyrosol were found to inhibit overall enzyme activity and TG synthesis. Overall, our findings provide new and valuable insights into the influence of reaction conditions on the stability and specificity of β-galactosidase. 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

N-(2-chloro-4-pyridyl)-N′-phenylurea (CPPU) and N-phenyl-1,2,3-thidiazole-5ylurea (TDZ) are plant growth regulators used for seedless treatment in grape. In this study, the flowers of ‘Shine Muscat’ (Vitis labruscana Bailey × V. vinifera L.) were treated with 3, 5, and 10 mg/L CPPU and TDZ one week before flowering. The results showed that both CPPU and TDZ treatments reduced the pollen germination rate and caused abnormal stamen and pollen grain phenotypes, resembling the male sterility observed in ‘Y_14’ (a novel grapevine germplasm derived from the self-progeny of ‘Shine Muscat’). Using RNA-seq technology, the stamens of flowers treated with 10 mg/L CPPU (CPPU_10), 10 mg/L TDZ (TDZ_10), and the control (CK) were analyzed. A total of 520 and 722 differentially expressed genes (DEGs) were identified in CPPU and TDZ treatments, respectively. GO and KEGG analyses revealed that the common pathways leading to pollen abortion in both treatments were primarily associated with hydrolase activity (acting on glycosyl bonds), phenylpropanoid biosynthesis, pentose and glucuronate interconversions, and ABC transporters. By comparing the DEGs across the three groups (Y_14 vs. SM, CPPU_10 vs. CK, TDZ_10 vs. CK), 16 DEGs exhibited similar expression patterns. Further tissue-specific expression analysis identified nine genes that were highly expressed in stamens and shared the same expression pattern in sterile lines. These findings provide a foundation for further studies on the impact of CPPU and TDZ treatments on grape stamen fertility. Full article

With the increasing scarcity of traditional hardwood sawdust resources, developing sustainable substrates for edible fungi cultivation has become an urgent industrial priority. This study systematically evaluated the effects of bamboo sawdust substitutions (20%, 30%, 40%, and 50%) on mycelial growth, fruiting body development, and nutritional quality of Auricularia heimuer, while elucidating the underlying molecular mechanisms through transcriptome sequencing. The results demonstrated that bamboo substitution of ≤30% maintained normal mycelial growth and fruiting body differentiation, with 20% and 30% substitutions increasing yields by 5.30% and 3.70%, respectively, compared to the control. However, 50% substitution significantly reduced yield by 9.49%. Nutritional analysis revealed that 20–40% bamboo substitution significantly enhanced the contents of crude protein, polysaccharides, and essential minerals (calcium, iron, and selenium) in fruiting bodies. Transcriptome analysis identified upregulation of glycosyl hydrolase family genes and downregulation of redox-related genes with increasing bamboo proportions. Biochemical assays confirmed these findings, showing decreased oxidative substances and increased reductive compounds in mycelia grown with high bamboo content, which indicate disrupted cellular redox homeostasis. This study provides both a practical solution to alleviate the “edible mushrooms derived from lignicolous fungi–forest conflict” and fundamental insights into fungal adaptation mechanisms to non-wood substrates, thus establishing a theoretical foundation for the valorization of agricultural and forestry wastes. 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

Background/Objectives: Obesity has reached pandemic proportions, with predictions suggesting that, by 2030, over 1.5 billion people will be affected. Pancreatic lipase (PL), the enzyme primarily responsible for the absorption of dietary lipids, presents a potential target for obesity management. However, while porcine pancreatic lipase (PPL) is commonly used as the enzyme source for screening potential inhibitors, its effect on human pancreatic lipase (HPL) is rarely reported. This work aimed to screen the inhibitory effects of a library of flavonoids with different functional groups on the activity of PL from the human pancreas (triacylglycerol acyl hydrolase, EC 3.1.1.3) and compare it to the effects of the porcine pancreas (type II, EC 3.1.1.3), establishing, whenever possible, a structure–activity relationship. Methods: The inhibitory effects of a library of 48 flavonoids with different hydroxy, glycosyl, rutinosyl, galloyl, and extended alkyl groups were evaluated against PPL and HPL. The kinetic parameters and inhibitory mechanisms of the most active flavonoids were determined, and in silico docking studies of the more potent flavonoids were also performed, using the active site of HPL. Results/Conclusions: Variations in enzyme catalytic activity were observed depending on the source of the enzyme. The inhibitory effect was particularly influenced by the presence of extended alkyl groups at the C-3 of the C-ring and the C2=C3 double bond of the C-ring and the presence of a pyrogallol group at the C-2′, C-3′ and C-4′ of the B-ring. Docking results showed a strong correlation between docking scores and observed inhibitory activities, highlighting the critical role of specific substituents on the flavonoid backbone in enhancing detailed interaction dynamics with key amino acids. Compounds 28, 29, and 30, with alkyl groups, showed the highest docking scores, interacting with residues HIS151, PHE215, ARG256, and HIS263. Further analysis also revealed that specific substituents improved pocket occupancy and formed additional interactions with residues TYR114, PRO180, ILE209, and PHE215, which are crucial for inhibition. These binding characteristics closely mimic those observed with orlistat, reinforcing their mechanistic similarities in inhibiting HPL and validating their inhibitory activities. Full article

OGP, encoded by the Ovgp1 gene, is the major non-serum oviductal protein in most mammals. In the genome of Rattus norvegicus, Ovgp1 has been identified as a pseudogene. However, Mus musculus presents a functional gene. As the rat and the mouse belong to the subfamily Murinae, Ovgp1 has probably been lost after their divergence. This study aims to determine when the pseudogenization event occurred and which proteins could replace its function. To attain that, the potential expression of members belonging to the GH18 family is investigated in the rat oviduct by means of molecular and proteomic analyses. Specific Ovgp1 regions are sequenced in different murine rodent species. The analysis reveals the presence of stop codons only in some species of the Rattini tribe, suggesting that the majority of the murine species present a functional gene. Thus, the pseudogenization of Ovgp1 could be dated back to around 10 Mya, after the divergence of the Rattini tribe. The expression of several genes and proteins of the GH18 family, such as Chia, Chit1, Chi3l1, and Chid1, are detected in the rat oviduct. This study opens the door for further research on GH18 family proteins that mimic the OGP functions in species where Ovgp1 is pseudogenized. Full article

Cold-adapted microorganisms possess cold-active enzymes with potential applications in different industries and research areas. In this study, two genes encoding β-d-galactosidases belonging to Glycoside Hydrolase families 2 and 42 from the psychrotolerant Arctic bacterium Arthrobacter sp. S3* were cloned, expressed in Escherichia coli and Komagataella phaffii, purified and characterized. The GH2 β-d-galactosidase is a tetramer with a molecular weight of 450 kDa, while the GH42 β-d-galactosidase is a 233 kDa trimer. The Bgal2 was optimally active at pH 7.5 and 22 °C and maintained 57% of maximum activity at 10 °C, whereas the Bgal42 was optimally active at pH 7.0 and 40 °C and exhibited 44% of maximum activity at 10 °C. Both enzymes hydrolyzed lactose and showed transglycosylation activity. We also found that 2 U/mL of the Bgal2 hydrolyzed 85% of lactose in milk within 10 h at 10 °C. The enzyme synthesized galactooligosaccharides, heterooligosaccharides, alkyl galactopyranosides and glycosylated salicin. The Bgal42 synthesized galactooligosaccharides and 20 U/mL of the enzyme hydrolyzed 72% of milk lactose within 24 h at 10 °C. The properties of Arthrobacter sp. S3* Bgal2 make it a candidate for lactose hydrolysis in the dairy industry and a promising tool for the glycosylation of various acceptors in the biomedical sector. Full article

Meloidogyne causes a devastating disease known as root-knot that affects tomatoes and other cash crops worldwide. Conversely, Paraburkholderia tropica has proven beneficial in mitigating the effects of various pathogens in plants. We aimed to unravel the molecular events that underlie the beneficial effects of the bacterium and the detrimental impacts of the nematode when inoculated separately or together in tomato plants. The transcriptional responses induced by P. tropica (TB group (tomato-bacteria group)), Meloidogyne spp. (TN group (tomato-nematode group)) or by the two agents (TBN group (tomato-bacteria-nematode group)) in tomato were assessed by RNA-seq. We implemented a transcript discovery pipeline which allowed the identification of 2283 putative novel transcripts. Differential expression analysis revealed that upregulated transcripts were much more numerous than downregulated ones. At the gene ontology level, the most activated term was ‘hydrolase activity acting on ester bonds’ in all groups. In addition, when both microbes were inoculated together, ‘hydrolase activity acting on O-glycosyl compounds’ was activated. This finding suggests defense responses related to lipid and carbohydrate metabolism, membrane remodeling and signal transduction. Notably, defense genes, transcription factors and protein kinases stood out. Differentially expressed transcripts suggest the activation of a multifaceted plant defense response against the nematode occurred, which was exacerbated by pre-inoculation of P. tropica. Full article

Autolysis in the sea cucumber Apostichopus japonicus is typically triggered by degradation caused by microorganisms within their bodies. However, information on this topic remains limited. Recently, we isolated and purified a bacterial strain from hydrolyzed instant sea cucumber samples. To investigate its potential role in the autolysis process, this study employed whole-genome sequencing and metabolomics to explore its genetic and metabolic characteristics. The identified strain was classified as Lysinibacillus xylanilyticus and designated with the number XL-2024. Its genome size is 5,075,210 bp with a GC content of 37.33%, encoding 5275 genes. Functional database comparisons revealed that the protein-coding genes were distributed among glucose metabolism hydrolase, metal hydrolase, lysozyme, cell wall hydrolase, and CAZymes. Compared to 20 closely related strains, L. xylanilyticus XL-2024 shared 1502 core homologous genes and had 707 specific genes. These specific genes were mainly involved in the carbohydrate metabolism pathway and exhibited glycosyl bond hydrolase activity. Metabolomic analysis showed that L. xlanilyticus XL-2024 produced several metabolites related to polysaccharide degradation, including peptidase, glucanase, and pectinase. Additionally, the presence of antibacterial metabolites such as propionic acid and ginkgo acid among its metabolites may enhance the stability of the sea cucumber hydrolysate. In summary, L. xylanilyticus XL-2024 may play a pivotal role in the autolysis of A. japonicus. The results of this study provide a strong foundation for understanding how to prevent autolysis in A. japonicus and for better utilizing L. xylanilyticus XL-2024. Full article

This study investigated the characterization of a novel multifunctional enzyme, RuXyn394, derived from the metagenome of beef cattle rumen, and its impact on the in vitro microbial fermentation of wheat straw. RuXyn394, a member of the glycosyl hydrolase 11 family, displayed optimal activity under diverse pH and temperature conditions: xylanase at pH 5.5 and 50 °C, acetyl esterase at pH 6.5 and 60 °C, exoglucanase at pH 7.0 and 50 °C, and endoglucanase at pH 6.0 and 50 °C. The enzyme’s xylanase, endoglucanase, and exoglucanase activities exhibited remarkable pH stability across the range of pH 3–8 and maintained a relatively stable performance at temperatures from 20 to 50 °C, 20 to 60 °C, and 20 to 70 °C, respectively. The xylanase function, with the highest k_cat/K_m ratio, was identified as the predominant activity of RuXyn394. The enzyme’s various functions responded uniquely to metal ions; notably, the addition of 5 mM K⁺ significantly boosted the activities of xylanase, exoglucanase, and endoglucanase by 55.5%, 53.5%, and 16.4%, respectively, without affecting its acetyl esterase activity. Over the course of three time points (30 min, 60 min, 120 min), the degradation products of wheat straw xylan, including xylopentaose, xylotetraose, xylotriose, xylobiose, xylose, and total xylooligosaccharides, constituted an average of 18.4%, 33.7%, 20.6%, 22.9%, 4.3%, and 95.7% of the total products, respectively. RuXyn394 effectively hydrolyzed wheat straw, resulting in augmented volatile fatty acid production and ammonia-N levels during in vitro microbial fermentation. These findings indicate the potential of RuXyn394 as a novel and highly efficient enzyme preparation, offering promising prospects for the valorization of wheat straw, an agricultural by-product, in ruminant diets. Full article