Search Results (100)

A series of new 3′-deoxyribosides of substituted benzimidazoles was obtained by the chemo-enzymatic method using genetically engineered E. coli purine nucleoside phosphorylase (PNP). In the case of asymmetrically substituted benzimidazole derivatives, a mixture of N1- and N3-regioisomers was formed (confirmed by NMR). The antiviral activity of the obtained compounds against herpes simplex virus 1 of reference strain L2 and a strain deeply resistant to acyclovir in Vero E6 cell culture was studied. 4,6-Difluoro-1-(β-D-3′-deoxyribofuranosyl)benzimidazole (IC₅₀ = 250.92 µM, SI = 12.00) and 4,5,6-trifluoro-1-(β-D-3′-deoxyribofuranosyl)benzimidazole (IC₅₀ = 249.96 µM, SI = 16.00) showed significant selective activity against both viral models in comparison to ribavirin (IC₅₀ = 511.88 µM, SI > 8.00). 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

Although enormous efforts have been made to prepare tasty and soluble steviol glycosides (SGs), the structure–property relationship of SGs still remains unclear, neither in experiment fact nor in the mechanism, such as the influence of linkage type and position of substituted glucosyl on physiochemical properties and sensory features of SGs. The favorable SGs, rebaudioside D (RD) and rebaudioside A (RA), possess good edulcorant quality, poor solubility, and other significantly different physical properties. This research chose two pairs of isomeric SGs, RA and its isomer rebaudioside E (RE) and RD and its isomer RA1G (a synthetic SG, α-1,6-mono-glucosylated RA), to conduct a comparative study, aiming to reveal the structure–property relevance on their solubility, sweetness, stability, and crystal structure. The RA1G presents an aqueous solubility 13 times that of RA and 137 times that of RD and exhibits better edulcorant quality than that of RA, similar to RD. The results indicate that the glucosyl linkage type and position have a stronger impact on the properties of the SGs than the number of glucosyl moieties. The underlying mechanism of their structure–property relevance was elucidated by analyzing the interaction energies between the SGs with solvent and human receptor proteins, respectively. 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

Cold-active enzymes hold promise for energy-efficient processes. Amylases are widely used in household and industrial applications, but only a few are cold-active. Here we describe three novel secreted amylases, Rho13, Ika2 and I3C6, all from bacteria growing in the cold and alkaline ikaite columns in Greenland. They all hydrolyzed starch to smaller malto-oligomers, but only Rho13 and Ika2 hydrolyzed cyclodextrins, and only Ika2 displayed transglycosylation activity. Ika2 forms a stable dimer, while both Rho13 and I3C6 are mainly monomeric. They all have optimal active temperatures around 30–35 °C and significant enzymatic activity below 20 °C, but Rho13 and I3C6 had an alkaline optimal pH, while Ika2 was markedly acidophilic. They showed complex dependence on Ca²⁺ concentration, with the activity of Rho13 and I3C6 following a bell-shaped curve and Ika2 being unaffected; however, removal of Ca²⁺ reduced the stability of all three enzymes. Loss of structure occurred well above the temperature of optimal activity, showing the characteristic psychrophilic divorce between activity and stability. MD simulations showed that Ika2 did not have a well-defined Ca²⁺ binding site, while Rho13 and I3C6 both maintained one stably bound Ca²⁺ ion. We identified psychrophilic features as higher levels of backbone fluctuations compared to mesophilic counterparts, based on a lower number of internal hydrogen bonds and salt bridges. This increased fluctuation was also found in regions outside the active site and may provide easier substrate access and accommodation, as well as faster barrier transitions. Our work sheds further light on the many ways in which psychrophilic enzymes adapt to increased catalysis at lower temperatures. Full article

Phenylethanoid glycosides (PhGs) are widely occurring secondary metabolites of medicinal plants with interesting biological activities such as antioxidant, anti-inflammatory, neuroprotective, antiviral, hepatoprotective, immunomodulatory, etc. They are characterized by a structural core formed by a phenethyl alcohol, usually tyrosol or hydroxytyrosol, attached to β-D-glucopyranose via a glycosidic bond. This core is usually further decorated by attached phenolic acids or another saccharide. Several studies suggest an important role of the saccharidic fragment in the biological activities of PhGs, provoking demand for new glycovariants of natural PhGs. This study presents the preparation of β-mannosylated analogs of tyrosol β-D-glucopyranoside (salidroside) and hydroxytyrosol β-D-glucopyranoside (hydroxysalidroside). While the chemical synthesis of β-D-mannopyranosides is rather challenging, they can be prepared by enzymatic catalysis. We found that Novozym 188, an industrial β-glucosidase, also contains β-mannosidase and used this enzyme in the preparation of tyrosol β-D-mannopyranoside and hydroxytyrosol β-D-mannopyranoside in 12 and 16% chemical yields, respectively, by transglycosylation from β-D-mannopyranosyl-(1→4)-D-mannose. The mannosylation was chemoselective and occurred exclusively on the primary hydroxyls of tyrosol and hydroxytyrosol, and the glycosylation of phenolic moieties of the aglycons was observed. 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

Sucrose phosphorylase (SPase) is a member of the glycoside hydrolase family 13, catalyzing the reversible phosphorolysis of sucrose to produce α–glucose–1–phosphate and exhibiting transglycosylation activity toward multiple substrates. Its wide substrate specificity enables the synthesis of various glycosides, which are broadly applied in food, cosmetics, and pharmaceuticals. However, the industrial application of SPase is constrained by its poor thermostability and limited transglycosylation activity. Therefore, current research focuses on enhancing the thermostability and transglycosylation activity of SPase through efficient engineering strategies based on its crystal structure and catalytic mechanism. This paper systematically reviews the crystal structure and catalytic mechanism of SPase, outlines the application of protein engineering and immobilization strategies in improving the thermostability of SPase, and analyzes how modifications at key amino acid sites affect the synthesis of typical glycosylation products. It also summarizes the limitations of SPase engineering modification strategies and explores the potential of diversified approaches for SPase modification, highlighting its broad application prospects in industrial production and laying a solid foundation for further advancements in SPase engineering modification and its industrial application. Full article

This study investigated the impact of a transglycosylated product (ACOD) catalyzed by Leuconostoc mesenteroides MKSR dextransucrase using sucrose as a glucosyl donor and both maltose and Artemisia capillaris as acceptors on gut microbiota through fecal fermentation. ACOD promoted the growth of probiotics such as Lactiplantibacillus plantarum, Lacticaseibacillus casei, Lacticaseibacillus rhamnosus GG, and Leuconostoc mesenteroides MKSR, while inhibiting the growth of pathogenic bacteria such as Escherichia coli, E. coli O157:H7, Enterococcus faecalis, Listeria monocytogenes, Staphylococcus aureus, Shigella flexneri, Streptococcus mutans, Pseudomonas aeruginosa, and Bacillus cereus during independent cultivation. Fecal fermentation for 24 h revealed that ACOD significantly increased the production of short-chain fatty acids (SCFAs) compared to the blank and fructoooligosaccharide (FOS) groups. Specifically, ACOD led to a 4.5-fold increase in acetic acid production compared to FOSs and a 3.3-fold increase in propionic acid production. Both the ACOD and FOS groups exhibited higher levels of butyric acid than the blank. Notably, ACOD significantly modulated the composition of the gut microbiota by increasing the relative abundances of Lactobacillus and decreasing Escherichia/Shigella and Salmonella. In contrast, FOSs remarkably promoted the growth of Salmonella. These findings suggest that ACOD is a potential candidate for prebiotics that improve the intestinal environment by being actively used by beneficial bacteria. Full article

Nucleoside phosphorylases (NPs) are pivotal enzymes in the salvage pathway, catalyzing the reversible phosphorolysis of nucleosides to produce nucleobases and α-D-ribose 1-phosphate. Due to their efficiency in catalyzing nucleoside synthesis from purine or pyrimidine bases, these enzymes hold significant industrial importance in the production of nucleoside-based drugs. Given that the thermodynamic equilibrium for purine NPs (PNPs) is favorable for nucleoside synthesis—unlike pyrimidine NPs (PyNPs, UP, and TP)—multi-enzymatic systems combining PNPs with PyNPs, UPs, or TPs are commonly employed in the synthesis of nucleoside analogs. In this study, we report the first development of two engineered bifunctional fusion enzymes, created through the genetic fusion of purine nucleoside phosphorylase I (PNP I) and thymidine phosphorylase (TP) from Thermus thermophilus. These fusion constructs, PNP I/TP-His and TP/PNP I-His, provide an innovative one-pot, single-step alternative to traditional multi-enzymatic synthesis approaches. Interestingly, both fusion enzymes retain phosphorolytic activity for both purine and pyrimidine nucleosides, demonstrating significant activity at elevated temperatures (60–90 °C) and within a pH range of 6–8. Additionally, both enzymes exhibit high thermal stability, maintaining approximately 80–100% of their activity when incubated at 60–80 °C over extended periods. Furthermore, the transglycosylation capabilities of the fusion enzymes were explored, demonstrating successful catalysis between purine (2′-deoxy)ribonucleosides and pyrimidine bases, and vice versa. To optimize reaction conditions, the effects of pH and temperature on transglycosylation activity were systematically examined. Finally, as a proof of concept, these fusion enzymes were successfully employed in the synthesis of various purine and pyrimidine ribonucleoside and 2′-deoxyribonucleoside analogs, underscoring their potential as versatile biocatalysts in nucleoside-based drug synthesis. Full article

Achieving enzymatic food processing at high substrate concentrations can significantly enhance production efficiency; however, related studies are notably insufficient. This study focused on the enzymatic synthesis of fructooligosaccharides (FOS) at high temperature and high substrate concentration. Results revealed that increased viscosity and limited substrate solubility in high-concentration systems could be alleviated by raising the reaction temperature, provided it aligned with the enzyme’s thermostability. Further analysis of enzyme thermostability in real sucrose solutions demonstrates that the enzyme’s thermostability was remarkedly improved at higher sucrose concentrations, evidenced by a 10.3 °C increase in melting temperature (T_m) in an 800 g/L sucrose solution. Building upon these findings, we developed a novel method for enzymatic FOS synthesis at elevated temperatures and high sucrose concentrations. Compared to existing commercial methods, the initial transglycosylation rate and volumetric productivity for FOS synthesis increased by 155.9% and 113.5%, respectively, at 65 °C in an 800 g/L sucrose solution. This study underscores the pivotal role of substrate concentration, incubation temperature, and the enzyme’s actual status in advancing enzyme-catalyzed processes and demonstrates the potential of enzymatic applications in enhancing food processing technologies, providing innovative strategies for the food industry. Full article

Sucrose phosphorylase (SPase), a member of the glycoside hydrolase GH13 family, possesses the ability to catalyze the hydrolysis of sucrose to generate α-glucose-1-phosphate and can also glycosylate diverse substrates, showcasing a wide substrate specificity. This enzyme has found extensive utility in the fields of food, medicine, and cosmetics, and has garnered significant attention as a focal point of research in transglycosylation enzymes. Nevertheless, SPase encounters numerous obstacles in industrial settings, including low enzyme yield, inadequate thermal stability, mixed regioselectivity, and limited transglycosylation activity. In-depth exploration of efficient expression strategies and molecular modifications based on the crystal structure and functional information of SPase is now a critical research priority. This paper systematically reviews the source microorganisms, crystal structure, and catalytic mechanism of SPase, summarizes diverse heterologous expression systems based on expression hosts and vectors, and examines the application and molecular modification progress of SPase in synthesizing typical glycosylated products. Additionally, it anticipates the broad application prospects of SPase in industrial production and related research fields, laying the groundwork for its engineering modification and industrial application. Full article

Enzymatic transglycosylation of the fleximer base 4-(4-aminopyridine-3-yl)-1H-pyrazole using recombinant E. coli purine nucleoside phosphorylase (PNP) resulted in the formation of “non-typical” minor products of the reaction. In addition to “typical” N1-pyrazole nucleosides, a 4-imino-pyridinium riboside and a N1-pyridinium-N1-pyrazole bis-ribose derivative were formed. N1-Pyrazole 2′-deoxyribonucleosides and a N1-pyridinium-N1-pyrazole bis-2′-deoxyriboside were formed. But 4-imino-pyridinium deoxyriboside was not formed in the reaction mixture. The role of thermodynamic parameters of key intermediates in the formation of reaction products was elucidated. To determine the mechanism of binding and activation of heterocyclic substrates in the E. coli PNP active site, molecular modeling of the fleximer base and reaction products in the enzyme active site was carried out. As for N1-pyridinium riboside, there are two possible locations for it in the PNP active site. The presence of a relatively large space in the area of amino acid residues Phe159, Val178, and Asp204 allows the ribose residue to fit into that space, and the heterocyclic base can occupy a position that is suitable for subsequent glycosylation. Perhaps it is this “upside down” arrangement that promotes secondary glycosylation and the formation of minor bis-riboside products. Full article

Glycogen, an α-glucan polymer serving as an energy storage compound in microorganisms, is synthesized through distinct pathways (GlgC-GlgA or GlgE pathway). Both pathways involve multiple enzymes, with a shared glycogen branching enzyme (GBE). GBEs play a pivotal role in establishing α-1,6-linkages within the glycogen structure. GBEs are also used for starch modification. Understanding how these enzymes work is interesting for both glycogen synthesis in microorganisms, as well as novel applications for starch modification. This study focuses on a putative enzyme GH13_9 GBE (PoGBE13), present in a polysaccharide utilization locus (PUL) of Pontibacter sp. SGAir0037, and related to the GlgE glycogen synthesis pathway. While the PUL of Pontibacter sp. SGAir0037 contains glycogen-degrading enzymes, the branching enzyme (PoGBE13) was also found due to genetic closeness. Characterization revealed that PoGBE13 functions as a typical branching enzyme, exhibiting a relatively high branching over non-branching (hydrolysis and α-1,4-transferase activity) ratio on linear maltooctadecaose (3.0 ± 0.4). Besides the GH13_9 GBE, a GH57 (PoGH57) enzyme was selected for characterization from the same PUL due to its undefined function. The combined action of both GH13 and GH57 enzymes suggested 4-α-glucanotransferase activity for PoGH57. The characterization of these unique enzymes related to a GlgE glycogen synthesis pathway provides a more profound understanding of their interactions and synergistic roles in glycogen synthesis and are potential enzymes for use in starch modification processes. Due to the structural similarity between glycogen and starch, PoGBE13 can potentially be used for starch modification with different applications, for example, in functional food ingredients. Full article

Fusarium is a genus that mostly consists of plant pathogenic fungi which are able to produce a broad range of toxic secondary metabolites. In this study, we focus on a type A trichothecene-producing isolate (15-39) of Fusarium sporotrichioides from Lower Austria. We assessed the secondary metabolite profile and optimized the toxin production conditions on autoclaved rice and found that in addition to large amounts of T-2 and HT-2 toxins, this strain was able to produce HT-2-glucoside. The optimal conditions for the production of T-2 toxin, HT-2 toxin, and HT-2-glucoside on autoclaved rice were incubation at 12 °C under constant light for four weeks, darkness at 30 °C for two weeks, and constant light for three weeks at 20 °C, respectively. The HT-2-glucoside was purified, and the structure elucidation by NMR revealed a mixture of two alpha-glucosides, presumably HT-2-3-O-alpha-glucoside and HT-2-4-O-alpha-glucoside. The efforts to separate the two compounds by HPLC were unsuccessful. No hydrolysis was observed with two the alpha-glucosidases or with human salivary amylase and Saccharomyces cerevisiae maltase. We propose that the two HT-2-alpha-glucosides are not formed by a glucosyltransferase as they are in plants, but by a trans-glycosylating alpha-glucosidase expressed by the fungus on the starch-containing rice medium. Full article