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Citrus is an important dietary source of flavonoid glycosides, and UDP-glycosyltransferases (UGTs) are the key enzymes responsible for their glycosylation. In this study, a genome-wide analysis of the CitUGT gene family was conducted to identify CitUGTs that contribute to flavonoid 4'- O -glucosides biosynthesis. Our analysis identified 136 CitUGTs in the Citrus clementina genome, classifying them into 18 phylogenetic groups (A-R) and 25 families. This classification was strongly supported by consistent gene structures and motif patterns. Moreover, we identified a CitUGT gene ( Ciclev10025462m , designated CitUGT72AZ4 ) that encodes flavonoid 4'- O -glucosyltransferase for the first time in citrus. This enzyme preferentially glycosylated the 4'-OH group of multiple flavonoids, exhibiting higher catalytic efficiency toward quercetin and three flavones in vitro . Virus-induced gene silencing of CitUGT72AZ4 significantly decreased the accumulation of flavonoid 4'- O -glucosides. These results indicated that CitUGT72AZ4 participated in the biosynthesis of flavonoid 4'- O -glucoside in citrus. Overall, our findings provide valuable insights into the CitUGT gene family and contribute to its functional characterization.

β-1,3 Glucan synthase (GS) is essential for fungal cell wall biosynthesis. The GS holoenzyme comprises the glycosyltransferase FKS1 and its regulatory factor Rho1, a small GTPase. However, the mechanism by which Rho1 activates FKS1 in a GTP-dependent manner remains unclear. Here, we present two cryo-EM structures of FKS1, apo and in complex with Rho1. FKS1 adopts a cellulose synthase-like conformation. The interaction between Rho1 and FKS1 is enhanced in the presence of GTPγS. Rho1 is positioned within a pocket between the glycosyltransferase domain of FKS1 (GT domain) and the transmembrane helix spanning TM7-15. Comparison of the two structures reveals extensive conformational changes within FKS1. These alterations suggest that Rho1's GTP/GDP cycling may act as a molecular pump, promoting a dynamic transition between the resting and active states of FKS1. Notably, Rho1 triggers FKS1 conformational changes that may push the growing glucan chain into FKS1's transmembrane channel, thereby facilitating β-1,3-glucan elongation., Competing Interests: Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Biflavonoids, a distinctive subclass of plant flavonoids, have a unique dimerized structure and possess a range of biological activities. The clinical applications of biflavonoids in human health have been impeded by challenges related to bioavailability and hydrophilicity. In contrast, biflavonoid glycosides, which demonstrate enhanced pharmacodynamic and pharmacokinetic properties compared to their aglycones, are notably limited in availability. In this work, we developed a robust enzymatic system to biosynthesize biflavonoid glycosides using O -glycosyltransferase UGT74AN2 and sucrose synthase At SuSy. This innovative system exhibited remarkable substrate promiscuity successfully, glycosylating 10 structurally diverse biflavonoids. Through purification and structural characterization, we identified four biflavonoid monoglycosides ( 1a , 2a , 4a , and 5a ) as well as two diglycosides ( 1b and 3b ). All synthesized products showed a significant increase in water solubility compared to their aglycones, with enhancements ranging from 20- to 980-fold. Furthermore, compound 1a demonstrated significantly enhanced antiproliferative activity against PC-3 cells compared to its corresponding aglycones. Metabolomic and transcriptomic analyses showed that the increased antitumor activity of 1a may be attributed to changes in the expression levels of various drug transporters, particularly within the ABC, PDE, and ATPase gene families. While compound 1 elevated the mRNA levels of several ABC transporters and ATPases, 1a did not induce these effects, highlighting a distinct mode of action. This study established an efficient enzymatic approach for the biosynthesis of biflavonoid glycosides and underscored their potential as valuable small molecules for drug discovery.

β-1,2-Glucans are physiologically important polymers for interactions such as symbiosis and pathogenesis between organisms and adaptation to environmental changes. However, rarity of β-1,2-glucans in nature limits exploration of related enzymes. Recently, many β-1,2-glucan-degrading enzymes have been found after identification of a novel phosphorylase acting on β-1,2-glucooligosaccharides. The expansion of the repertoire has reached revelation of the cyclization mechanism of cyclic β-1,2-glucan synthase and led to finding of new enzymes catalyzing cyclization of β-1,2-glucans in a manner different from cyclic β-1,2-glucan synthase. In this review, we mainly focus on newly found enzymes that catalyze cyclization of β-1,2-glucans along with existence of β-1,2-glucan-associated carbohydrates in nature and introduction of the repertoire of β-1,2-glucan-degrading enzymes. KEY POINTS: • Newly found domain which cyclizes β-1,2-glucan created a new glycoside hydrolase family. • Cyclization is performed with a unique mechanism. • α-1,6-Cyclized β-1,2-glucan is produced by an enzyme in another newly found family., Competing Interests: Declarations. Ethics approval and consent to participate: Not applicable. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Steviol glycosides (SGs) are highly valued for their sweetness, safety, and zero calories, but their bitter taste and low solubility limit their application. Modifying glycosyl units is a promising strategy to improve sensory qualities. In this study, we identified the enzyme UGT94E13 through phylogenetic analysis and enzyme screening, which catalyzes the glycosylation of rebaudioside M2 (Reb M2) at the C-13 position, producing the novel β-1,6- O -glycosylated product rebaudioside M9 (Reb M9). Subsequently, the catalytic activity of UGT94E13 toward Reb M2 was enhanced 12.0-fold through a structure-guided enzyme engineering strategy, and the mechanism behind this enhancement was analyzed. Finally, an enzymatic cascade system comprising the optimal mutant UGT94E13-F169A/I185A and sucrose synthase At SuSy was constructed and optimized, achieving efficient synthesis of Reb M9 with a yield of 98.3% and a titer of 42.8 g·L -1 . Overall, this study provides an effective method for enhancing glycosylation of SGs and a reference for the glycosylation modification of natural products.

Enzymatic glycosylation is an efficient and biocompatible approach to enhance natural product bioavailability. Cellobiose phosphorylase, a novel glycosyltransferase, utilizes 1-phospho-glucose (1-p-Glc) as a glycosyl donor for regioselective glycosylation of various natural substrates. However, the high cost of 1-p-Glc limits the economic feasibility of the process. Thus, a dual-enzyme cascade system involving cellulase AcCel9A and cellobiose phosphorylase CbCBP using a co-immobilization strategy was developed to overcome this challenge. The system utilizes low-cost carboxymethyl cellulose (CMC) for continuous 1-p-Glc production, which was then used in the fluorodeoxy glucose (FDG) glycosylation to generate fluorodeoxy cellobiose (FDC), which potentially traces fungal infections. The compartmentalized co-immobilization of the two enzymes within the internal and external regions of a porous zeolitic imidazolate framework-8 (ZIF-8) carrier enhanced the overall stability of the dual-enzyme system. The immobilized enzymes retained approximately 63.3 % activity after seven reuse cycles and 74 % catalytic efficiency after 12 days of storage at room temperature. Therefore, the developed co-immobilized multi-enzyme system holds significant potential for industrial biocatalysis applications., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

The one-step deconstruction of sucrose into uridine 5'-diphosphate-glucose (UDP-Glc), an important sugar donor for transglycosylation, employing sucrose synthase (Susy) is emerging as a valuable sucrose utilization process. The insufficient activity and stability of Susy limit the productivity of UDP-Glc from sucrose. Here, an engineered Susy (Susy M6 ) that counteracted the activity-stability trade-off was developed with the half-life time and activity being 43-fold and 1.4-fold of wild-type, respectively. Tighter hydrophobic patches and stabilization of the SSN2 domain contributed to greater activity and stability. The use of Susy M6 in UDP-Glc production resulted in a satisfactory space-time yield of 73 g/L/h within 1 h. The cascade of different biocatalysts with Susy M6 , focusing on utilizing two products of sucrose decomposition, fructose and UDP-Glc, expanded sucrose utilization, efficiently promoting the UDP-Glc productivity and giving a cost-effective method for UDP-galactose (UDP-Gal) synthesis. This study demonstrated promising green pathways for producing multiple value-added products from sucrose using Susy., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

Cyclodextrin-glucanotransferase (CGTase, EC 2.4.1.19) is a multifunctional enzyme that catalyzes many enzymatic reactions including cyclization, binding, disproportionation and hydrolysis reactions, playing an important role in the enzymatic synthesis of compounds that are widely used in agriculture, pharmaceuticals, food, chemical and biotechnology industries. The present research is aimed to optimize the purification protocol for the extracellular CGTase of alkaliphilc bacterial strain Caldalkalibacillus mannanilyticus IB-OR17-B1 guaranteeing the enzyme homogeneity and its high yield. The improved combination of ultrafiltration and corn-starch (5% w/v) affinity sorption techniques resulted to mild and rapid isolation of electrophoritically homogenic enzyme at 18 × increase of its specific activity and yield 56%. The developed two-step procedure instead the practiced tree-step one using commonly ion-exchange chromatography as final purification technique highly contributes in advance of cost-effectiveness for industrial production and isolation of valuable CGTases.

The pathogen-associated C -glucosyltransferase IroB is involved in the biosynthesis of salmochelins, C -glucosylated derivatives of enterobactin (Ent), which is a triscatecholate siderophore of enteric bacteria including Salmonella enterica and Escherichia coli . Here, we reassess the ability of IroB to C -glucosylate non-native triscatecholate mimics of Ent, which may have utility in the design and development of siderophore-based therapeutics and diagnostics. We establish TRENCAM (TC) and MECAM (MC), synthetic Ent analogs with tris(2-aminoethyl)amine- or mesitylene-derived backbones replacing the trilactone core of Ent, respectively, and their monoglucosylated congeners as substrates of IroB. Time course analyses and steady-state kinetic studies, which were performed under conditions that provide enhanced activity relative to prior studies, inform the substrate selectivity and catalytic efficiencies of this enzyme. We extend these findings to the preparation of a siderophore-antibiotic conjugate composed of monoglucosylated TC and ampicillin (MGT-Amp). Examination of its antibacterial activity and receptor specificity demonstrates that MGT-Amp targets pathogenicity because it shows specificty for the pathogen-associated outer membrane receptor IroN. Overall, our findings extend the biochemical characterization of IroB and its substrate scope and illustrate the ability to leverage a bacterial C -glucosyltransferase for non-native chemoenzymatic transformations along with potential applications of salmochelin mimics.

To improve paste stability of cassava starch, including acid resistance, high-temperature shear resistance and freeze-thaw stability, cassava starch was modified by sequential maltogenic amylase and transglucosidase to form an optimally denser structure, or branched density (12.76 %), molecular density (15.17 g/mol/nm 3 ), and the proportions of short-branched chains (41.41 % of A chains and 44.01 % of B1 chains). Viscosity stability (88.52 %) of modified starch was higher than that (64.92 %) of native starch. After acidic treatment for 1 h, the viscosity of modified starch and native starch decreased by 56.53 % and 65.70 %, respectively. Compared to native starch, modified starch had lower water loss in freeze-thaw cycles and less viscosity reduction during high-temperature and high-shear processing. So, the appropriate molecular density and denser molecule structure enhanced paste stabilities of modified starch. The outcome expands the food and non-food applications of cassava starch., Competing Interests: Declaration of competing interest We confirm that there are no any conflicts of interest with respect to this publication and there has been no financial support for this article that could influence the outcome., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Amylosucrase (ASase; E.C. 2.4.1.4), a member of glycoside hydrolase family 13 (GH13), produces α-1,4-glucans and sucrose isomers using sucrose as its sole substrate. This study identifies and characterizes the dimeric structure of ASase from Deinococcus geothermalis (DgAS), highlighting essential amino acid residues for maintaining the dimeric state. The monomeric form, DgAS R30A, exhibited a higher affinity for sucrose compared to the wild-type (WT), especially during the formation of the ASase-glucose intermediate complex and subsequent hydrolysis. Notably, DgAS R30A produced a higher proportion of α-glucans with a degree of polymerization (DP) of ≤20 and fewer α-glucans with a DP of ≥31. This suggested that the reduced surface area of the oligosaccharide binding site in the monomeric form led to decreased binding of longer-chain maltooligosaccharides, favoring the formation of shorter DP α-glucans. Kinetic analysis revealed significantly lower Michaelis constants (K m ) for DgAS R30A's total and hydrolysis activities, with the overall performance (k cat /K m ) values for DgAS R30A exceeded those of the WT at all sucrose concentrations. Here, we report the first high-resolution homodimeric DgAS structure, revealing conserved active site residues and a unique dimerization interface. This study enhances our understanding of the molecular factors influencing the oligomeric state and enzyme activities., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

2-O-α-d-glucopyranosyl-l-ascorbic acid (AA-2G) is often substituted for l-ascorbic acid (L-AA) in health- and skincare products due to its enhanced stability and comparable antioxidant. Enzymatic catalysis for AA-2G is gaining widespread interest and sucrose phosphorylases (SPase) have shown promise in achieving higher yields. To enhance AA-2G synthesis, we screened and identified the SPase from Bifidobacterium longum (BlSPase) as the starting enzyme, with an optimal pH of 5.4 and temperature of 45 °C for AA-2G synthesis. Subsequently, we conducted semi-rational design on BlSPase based on modeling and molecular docking. L-AA was docked with the BlSPase model to analyze key residues in the 'loop-door' domain and substrate-binding pocket. Through alanine scanning and saturation mutagenesis, a mutant library was created. After single-point and composite mutation, L341V/V346P was identified as the optimal variant. Ultimately, within 72 h, we achieved yield of 358.6 g/L AA-2G with a molar conversion of L-AA reaching 75.7 %., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Decades of research on the infamous antinutritional steroidal glycoalkaloids (SGAs) in Solanaceae plants have provided deep insights into their metabolism and roles. However, engineering SGAs in heterologous hosts has remained a challenge. We discovered that a protein evolved from the machinery involved in building plant cell walls is the crucial link in the biosynthesis of SGAs. We show that cellulose synthase-like M [GLYCOALKALOID METABOLISM15 (GAME15)] functions both as a cholesterol glucuronosyltransferase and a scaffold protein. Silencing GAME15 depletes SGAs, which makes plants more vulnerable to pests. Our findings illuminate plant evolutionary adaptations that balance chemical defense and self-toxicity and open possibilities for producing steroidal compounds in heterologous systems for food, cosmetics, and pharmaceuticals.

The glycosylation of an alcohol group from a sugar nucleotide substrate involves proton release, so the reaction is favored thermodynamically at high pH. Here, we explored expansion of the alkaline pH range of sucrose synthase (SuSy; EC 2.4.1.13) to facilitate enzymatic glycosylation from uridine 5'-diphosphate (UDP)-glucose. The apparent equilibrium constant of the SuSy reaction (UDP-glucose + fructose ↔ sucrose + UDP) at 30 °C increases by ∼4 orders of magnitude as the pH is raised from 5.5 to 9.0. However, the SuSy in solution loses ≥80 % of its maximum productivity at pH ∼7 when alkaline reaction conditions (pH 9.0) are used. We therefore immobilized the SuSy on nanocellulose-based biocomposite carriers (∼48 U/g carrier; ≥ 50 % effectiveness) and reveal in the carrier-bound enzyme a substantial broadening of the pH-productivity profile to high pH, with up to 80 % of maximum capacity retained at pH 9.5. Using reaction by the immobilized SuSy with automated pH control at pH ∼9.0, we demonstrate near-complete conversion (≥ 96 %) of UDP-glucose and fructose (each 100 mM) into sucrose, as expected from the equilibrium constant (K eq = ∼7 × 10 2 ) under these conditions. Collectively, our results support the idea of glycosyltransferase-catalyzed synthetic glycosylation from sugar nucleotide donor driven by high pH; and they showcase a marked adaptation to high pH of the operational activity of the soybean SuSy by immobilization., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The author is an Editorial Board Member/Editor-in-Chief/Associate Editor/Guest Editor for [Journal name] and was not involved in the editorial review or the decision to publish this article., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Turanose (α-d-glucopyranosyl-(1→3)-α-d-fructose) is a rare disaccharide that is a potential low-calorigenic sweetener. This novel sucrose isomer has been efficiently synthesized by the amylosucrase from Bifidobacterium thermophilum (BtAS). In this study, we aimed to enhance turanose biosynthesis by designing a BtAS variant (BtAS-G374S) with improved thermal stability. The BtAS variant was immobilized on porosity-controlled silica carrier, and its enzymatic properties were thoroughly investigated. Using response surface methodology with central composite design, optimal immobilization conditions were determined to significantly boost the biosynthetic efficiency. The BtAS-G374S showed 1.6-fold higher specific activity (2.2 U/mg) than the wild-type enzyme (1.4 U/mg). Additionally, the turanose production yield of BtAS-G374S was significantly enhanced, reaching 65 %, compared to 25 % for the wild-type enzyme when reacting with 2 M sucrose. Immobilization of BtAS-G374S was optimized on controlled porosity carrier (CPC) silica carrier using Response Surface Methodology, achieving an enzyme activity of 7.89 U and an immobilization efficiency of 68.98 % under optimal conditions. Immobilization of BtAS-G374S on CPC silica carriers enhanced its pH and thermal stability. The immobilized enzyme showed a half-life of 50.23 h at 55 °C and retained 68 % of its initial biosynthetic yield after 10 reuses. These properties suggest its potential for efficient industrial turanose production., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

γ-Cyclodextrin (γ-CD) is an attractive material among the natural cyclodextrins owing to its excellent properties. γ-CD is primarily produced from starch by γ-cyclodextrin glycosyltransferase (γ-CGTase) in a controlled system. However, difficulty in separation and low conversion rate leads to high production costs for γ-CD. In this study, γ-CGTase from Bacillus sp. G-825-6 STB17 was used in γ-CD production from cassava starch. With the introduction of sodium tetraphenylborate (NaBPh 4 ), the total conversion rate was promoted from an initial 18.07 % to 50.49 % and the γ-CD ratio reached 78.81 % with a yield of 39.79 g/L. Furthermore, the mechanism was conducted via the determination of binding constant, which indicated that γ-CD exhibited much stronger binding strength with NaBPh 4 than β-CD. The reformation of water molecules and the chaotropic effect might be the main driving forces for the interaction. Additionally, the conformations of CD complexes were depicted by NMR and molecular docking. The results further verified different binding patterns between CDs and tetraphenylborate ions, which might be the primary reason for the specific binding. This system not only guides γ-CD production with an efficient and easy-to-remove production aid but also offers a new perspective on the selection of complexing agents in CD production., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Traditional metal-organic frameworks (MOFs) preparation is generally time-consuming, polluting, and lacking specificity for enzyme immobilization. This paper introduced a facile, rapid, and green method to produce three MOFs subsequently employed to purify and coimmobilize recombinant glycosyltransferase (UGT) and recombinant sucrose synthetase (SUSy) using histidine tag (His-tag) for the specific adsorption of Ni 2+ and Co 2+ from MOFs. This method simplified enzyme purification from crude extracts and enabled enzymes to be reused. The results demonstrated that NiCo-MOF exhibited a higher enzyme load (115.9 mg/g) than monometallic MOFs. Additionally, the NiCo-MOF@UGT&SUSy demonstrated excellent stability and efficiently produced the rare ginsenoside Rh2 by catalyzing a coupling reaction (95.6 μg/mL), solving the problem of the substrate cost of uridine diphosphate glucose (UDPG). The NiCo-MOF@UGT&SUSy retained 68.97% of the initial activity after 10 cycles. Finally, molecular docking studies elucidated the conversion mechanism of the target product Rh2. This technique is important in the industrialization of ginsenoside production and enzyme purification.

The study aimed to develop an efficient bioprocess for the discovery and synthesis of theanderose by using amylosucrase from Deinococcus deserti ( Dd AS). An unknown trisaccharide produced by Dd AS was detected by high-performance anion-exchange chromatography-pulsed amperometric detection and high-performance liquid chromatography-evaporative light scattering detection, purified using medium-pressure liquid chromatography, and identified as theanderose (α-d-glucopyranosyl-(1→6)-α-d-glucopyranosyl-(1→2)-β-d-fructofuranoside) through nuclear magnetic resonance and mass spectrometry. Dd AS synthesized theanderose with a 25.4% yield (174.1 g/L) using 2.0 M sucrose at 40 °C for 96 h. In an in vitro digestion model, theanderose showed a 6.5% hydrolysis rate over 16 h. Prebiotic efficacy tests confirmed that theanderose significantly enhanced the proliferation of selected Bifidobacterium strains in the culturing medium with theanderose as the main carbon source. Subsequently, fecal fermentation was performed by adding theanderose to the feces of 20 individuals of varying ages to assess its effect on the gut microbiota. Theanderose increased the relative abundance of Bifidobacteriaceae and Prevotellaceae while decreasing the population ratio of Lachnospiraceae and Ruminococcaceae . Conclusively, theanderose displayed excellent prebiotic potential when judged by low digestibility and selective growth of beneficial microbes over harmful microbes.

The effect of delignification on the adsorption capacity of loofah sponge-based immobilized metal affinity chromatography adsorbents was investigated with recombinant His-tagged trehalose synthase as the model protein. Pretreatments with [EMIM][Ac] ionic liquid at 80 °C for 5 h and with sodium chlorite/acetic acid at 80 °C for 2 h were found effective for the removal of lignin, leading to a loss in biomass of 15.7% and 25.2%, respectively. Upon delignification, the metal chelating capacities of the loofah sponge-based adsorbents prepared with 5-h ionic liquid pretreatment (712 ± 82 μmole Cu(II)/g) and with 2-h sodium chlorite/acetic acid pretreatment (1012 ± 18 μmole Cu(II)/g) were 38% and 97% higher than that of the control (514 ± 55 μmole Cu(II)/g), adsorbent prepared with untreated loofah sponge, respectively. Results of protein adsorption study indicated that the Co(II)-loaded adsorbent prepared with 2-h sodium chlorite/acetic acid pretreatment exhibited the highest adsorption capacity and selectivity for the recombinant His-tagged trehalose synthase, giving a purification product with a specific activity of 7.62 U/mg protein. The predicted maximum adsorption capacity of the delignified loofah sponge-based adsorbent, 2.04 ± 0.14 mg/g, was 73% higher than that of the control., (Copyright © 2024 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

2-O-(α-d-glucopyranosyl)-sn-glycerol (2-αGG) has been applied in the food industry due to its numerous physiological benefits. The synthesis of 2-αGG can be achieved through a cascade catalytic reaction involving sucrose phosphorylase (SP) and 2-O-α-glucosylglycerol phosphorylase (GGP). However, the low substrate transfer rates between free enzymes have hindered the efficiency of 2-αGG synthesis. To address this issue, a novel technology was developed to prepare sequential multi-enzyme nanoflowers via chemical crosslinking and protein assembly, thus overcoming diffusion limitations. Specifically, spatially sequential co-immobilized enzymes, referred to as SP-GGP@Cap, were created through the targeted assembly of Bifidobacterium adolescentis SP and Marinobacter adhaerens GGP on Ca 2+ . This assembly was facilitated by the spontaneous protein reaction between SpyTag and SpyCatcher. Compared to free SP-GGP, SP-GGP@Cap demonstrated improved thermal and pH stability. Moreover, SP-GGP@Cap enhanced the biosynthesis of 2-αGG, achieving a relative concentration of 98 %. Additionally, it retained the ability to catalyze the substrate to yield 61 % relative concentration of 2-αGG even after ten cycles of recycling. This study presents a strategy for the spatially sequential co-immobilization of multiple enzymes in a confined environment and provides an exceptional biocatalyst for the potential industrial production of 2-αGG., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Several lactic acid bacteria are capable of producing water-soluble exopolysaccharides such as dextran from sucrose by using glucansucrases. Several recombinant glucansucrases were described, however, yields were often limited and most dextrans were branched at position O3. In this study, the dextransucrase from Ligilactobacillus animalis TMW 1.971 was recombinantly produced without its N-terminal variable region and used for dextran synthesis. The enzyme expressed well and showed very high total as well as transferase activities compared to other glucansucrases. It was able to transfer nearly all glucose from sucrose to oligo- and polymeric products under certain conditions (about 95 % of glucose transferred). The high efficiency of the enzyme made it possible to obtain absolute dextran yields of up to 214.9 g/L from a 1.5 M sucrose solution. Structural characterization of the products showed that the dextrans produced have a rather low molecular weight, a narrow size distribution, and are completely linear. Furthermore, we showed that various low molecular weight dextrans or 1,6-linked isomalto-oligosaccharides can be efficiently produced by acid hydrolysis. Overall, we demonstrated that Ligilactobacillus animalis TMW 1.971 dextransucrase can be used to efficiently synthesize dextrans with a quite unique structural composition. The dextrans produced have a high potential for further applications such as synthesis of copolymers or size standards with a very defined molecular structure., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Glycosylation is an effective way to promote the total intake of polyphenols in humans by increasing the solubility of polyphenols. In this study, an efficient glycosylation system was built via the dynamic complexation of CD with polyphenols and synchronous coupling reaction with cyclodextrin glycosyltransferase (CGTase) in water. The glycosylation efficiencies of quercetin, naringenin, rutin, resveratrol and caffeic acid were 20.9, 3.6, 2.7, 3.4 and 1.5 times higher than the non-complexed system. To quantify conversion rate and determine the rate-limiting step, the mixed product was treated with amyloglucosidase to obtain α-glucosyl rutin, which was identified as rutin 4"-O-α-D-glucopyranoside with purity of 93.6 % and yield of 34.8 % from NMR, MS and HPLC analysis. The results of half-reaction kinetics showed that the catalytic efficiencies of ring-opening of γ-CD (k 1 ) and glycosylation reaction of rutin (k 2 ) were 621.92 and 9.43 mM -1 ·s -1 . The rate-limiting step was clarified for the first time, showing that the ring-opening ability of CGTase to CD was much higher than its glycosylation ability to polyphenols. It is speculated that the rapid ring-opening reaction of CD affected its dynamic complexation, releasing many polyphenols which were not utilized by CGTase in time. Therefore, adjusting the ratio and concentration of CD resulted in an optimal glycosylation molar yield of 84.1 % for rutin, which was the highest yield reported so far in water. This study established a universal system and clarified the rate-limiting step in the enzymatic glycosylation, providing theoretical guidance for efficient production of polyphenol glycosylation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

In tomato, sugar content is highly correlated with consumer preferences, with most consumers preferring sweeter fruit 1-4 . However, the sugar content of commercial varieties is generally low, as it is inversely correlated with fruit size, and growers prioritize yield over flavour quality 5-7 . Here we identified two genes, tomato (Solanum lycopersicum) calcium-dependent protein kinase 27 (SlCDPK27; also known as SlCPK27) and its paralogue SlCDPK26, that control fruit sugar content. They act as sugar brakes by phosphorylating a sucrose synthase, which promotes degradation of the sucrose synthase. Gene-edited SlCDPK27 and SlCDPK26 knockouts increased glucose and fructose contents by up to 30%, enhancing perceived sweetness without fruit weight or yield penalty. Although there are fewer, lighter seeds in the mutants, they exhibit normal germination. Together, these findings provide insight into the regulatory mechanisms controlling fruit sugar accumulation in tomato and offer opportunities to increase sugar content in large-fruited cultivars without sacrificing size and yield., Competing Interests: Competing interests: A patent on the use of SlCDPK27 and SlCDPK26 to improve fruit sugar content has been filed by S.H., J.Z., H.L., J.C. and G.Z. (CN202211616622.8; PCT/CN2023/138699). The other authors declare no competing interests., (© 2024. The Author(s).)

Enzymatically modified isoquercitrin (EMIQ) is a glyco-chemically modified flavonoid exhibiting notably high biological activity, such as antioxidant, anti-inflammatory and anti-allergic properties. However, the utilization of expensive substrates such as isoquercitrin and cyclodextrin in the conventional approach has hindered the industrial-scale production of EMIQ due to high cost and low yields. Hence, the development of a cost-effective and efficient method is crucial for the biological synthesis of EMIQ. In this study, a natural cascade catalytic reaction system was constructed with α-L-rhamnosidase and amylosucrase using the inexpensive substrates rutin and sucrose. Additionally, a novel approach integrating gradient temperature regulation into biological cascade reactions was implemented. Under the optimal conditions, the rutin conversion reached a remarkable 95.39% at 24 h. Meanwhile, the productivity of quercetin-3-O-tetraglucoside with the best bioavailability reached an impressive 41.69%. This study presents promising prospects for future mass production of EMIQ directly prepared from rutin and sucrose., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in the paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Here, we enzymatically produced a novel α-1,2-glucan, glucosylsucrose, that has a chemical structure significantly different from that of other glucans. This structural difference suggests its potential to modulate new physiological activities compared to known glucans. The enzyme TeGSS catalyzes the synthesis of this α-1,2-glucan from sucrose and UDP-glucose (UDPG). Using NMR spectroscopy, we elucidated the chemical structures of TeGSS-synthesized glucosylsucrose tri-, tetra-, and penta-saccharides in which the monosaccharide units are linked by α-1,2-glycosidic bonds. We also report the crystal structures of TeGSS co-crystallized with UDP and glucosylsucrose tri- and tetra-saccharides. Site-directed mutagenesis of residues in and around the TeGSS catalytic center has allowed us to propose a concerted S N i mechanism of action. Finally, we developed an enzyme-coupled reaction involving TeGSS and SuSyAc that allows production of UDPG for the synthesis of α-1,2-glucan., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Chalcones, including 4'-hydroxychalcones, have garnered significant attention in the area of drug discovery due to their diverse pharmacological properties, such as anti-inflammatory, antioxidative, and anticancer effects. However, their low water solubility and bioavailability limit their efficacy in vivo . Glycosylation presents a promising approach to enhance the water solubility, stability, and metabolic properties of chalcones. This study investigates the enzymatic glycosylation of eight chemically synthesized 4'-hydroxychalcones using a diverse set of sugar glucosyltransferases from bacterial, plant, and fungal sources, alongside Glycine max sucrose synthase (GmSuSy) in a cascade reaction. Among the tested enzymes, five exhibited a remarkable versatility for glycoside production, and for large-scale biotransformation, flavonoid 7- O -glucosyltransferase Sbaic7OGT from Scutellaria baicalensis was selected as the most effective. As a result of the experiments conducted, eight trans -chalcone glycosides were obtained. During the purification of the reaction products, we also observed the isomerization of the products by simple sunlight exposure, which resulted in eight additional cis -chalcone glycosides. This study highlights the novel use of a cascade reaction involving Glycine max sucrose synthase (GmSuSy) for the efficient glycosylation of trans -4'-hydroxychalcones, alongside the unexpected discovery of cis -chalcone glycosides during the purification process., Competing Interests: The authors declare no conflicts of interest.

The microdomains of plasmodesmata, specialized cell-wall channels responsible for communications between neighboring cells, are composed of various plasmodesmata-located proteins (PDLPs) and lipids. Here, we found that, among all PDLP or homologous proteins in Arabidopsis thaliana genome, PDLP5 and PDLP7 possessed a C-terminal sphingolipid-binding motif, with the latter being the only member that was significantly upregulated upon turnip mosaic virus and cucumber mosaic virus infections. pdlp7 mutant plants exhibited significantly reduced callose deposition, larger plasmodesmata diameters, and faster viral transmission. These plants exhibited increased glucosidase activity but no change in callose synthase activity. PDLP7 interacted specifically with glucan endo-1,3-β-glucosidase 10 (BG10). Consistently, higher levels of callose deposition and slower virus transmission in bg10 mutants were observed. The interaction between PDLP7 and BG10 was found to depend on the presence of the Gnk2-homologous 1 (GnK2-1) domain at the N terminus of PDLP7 with Asp-35, Cys-42, Gln-44, and Leu-116 being essential. In vitro supplementation of callose was able to change the conformation of the GnK2-1 domain. Our data suggest that the GnK2-1 domain of PDLP7, in conjunction with callose and BG10, plays a key role in plasmodesmata opening and closure, which is necessary for intercellular movement of various molecules., (Copyright © 2024 Science China Press. Published by Elsevier B.V. All rights reserved.)

Crocus sativus L. is a both medicinal and food bulbous flower whose qualities are geographically characterized. However, identification involving different places of origin of such substances is currently limited to single-omics mediated content analysis. Integrated metabolomics and proteomics, 840 saffron samples from six countries (Spain, Greece, Iran, China, Japan, and India) were analyzed using the QuEChERS extraction method. A total of 77 differential metabolites and 14 differential proteins were identified. The limits of detection of the method were 1.33 to 8.33 μg kg -1 , and the recoveries were 85.56% to 105.18%. Using homology modeling and molecular docking, the Gln84, Lys195, Val182 and Pro184 sites of Crocetin glucosyltransferase 2 were found to be the targets of crocetin binding. By multivariate statistical analysis (PCA and PLS-DA), different saffron samples were clearly distinguished. The results provided the basis for the selection and identification of high quality saffron from different producing areas., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The authors declare no conflicting financial interest., (Copyright © 2023. Published by Elsevier Ltd.)

Assessing the number of glucan chains in cellulose microfibrils (CMFs) is crucial for understanding their structure-property relationships and interactions within plant cell walls. This Review examines the conclusions and limitations of the major experimental techniques that have provided insights into this question. Small-angle X-ray and neutron scattering data predominantly support an 18-chain model, although analysis is complicated by factors such as fibril coalescence and matrix polysaccharide associations. Solid-state nuclear magnetic resonance (NMR) spectroscopy allows the estimation of the CMF width from the ratio of interior to surface glucose residues. However, there is uncertainty in the assignment of NMR spectral peaks to surface or interior chains. Freeze-fracture transmission electron microscopy images show cellulose synthase complexes to be "rosettes" of six lobes each consistent with a trimer of cellulose synthase enzymes, consistent with the synthesis of 18 parallel glucan chains in the CMF. Nevertheless, the number of chains in CMFs remains to be conclusively demonstrated.

This study aims to develop an efficient chitin-based purification system, leveraging a novel design where the target proteins, superfolding green fluorescent protein (sfGFP) and Thermus antranikianii trehalose synthase (TaTS), fused with a chitin-binding domain (ChBD) from Bacillus circulans WL-12 chitinase A1 and a tobacco etch virus protease (TEVp) cleavage site. This configuration allows for the effective immobilization of the target proteins on chitin beads, facilitating the removal of endogenous proteins. A mutant TEVp, H-TEVS219V-ChBD, fused with the His-tag and ChBD, is employed to cleave the target proteins from the chitin beads specifically. Subsequently, fresh chitin beads are added for adsorption to remove H-TEVS219V-ChBD in the solution, thereby significantly improving the purity of the target protein. Our results confirm that this system can efficiently and specifically purify and recover sfGFP and TaTS, achieving electrophoretic-grade purity exceeding 90%. This system holds significant potential for industrial production and other applications.

Polyols, or sugar alcohols, are widely used in the industry as sweeteners and food formulation ingredients, aiming to combat the incidence of diet-related Non-Communicable Diseases. Given the attractive use of Generally Regarded As Safe (GRAS) enzymes in both academia and industry, this study reports on an optimized process to achieve polyols transglucosylation using a dextransucrase enzyme derived from Leuconostoc mesenteroides . These enzyme modifications could lead to the creation of a new generation of glucosylated polyols with isomalto-oligosaccharides (IMOS) structures, potentially offering added functionalities such as prebiotic effects. These reactions were guided by a design of experiment framework, aimed at maximizing the yields of potential new sweeteners. Under the optimized conditions, dextransucrase first cleared the glycosidic bond of sucrose, releasing fructose with the formation of an enzyme-glucosyl covalent intermediate complex. Then, the acceptor substrate (i.e., polyols) is bound to the enzyme-glucosyl intermediate, resulting in the transfer of glucosyl unit to the tested polyols. Structural insights into the reaction products were obtained through nuclear maneic resonance (NMR) and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) analyses, which revealed the presence of linear α(1 → 6) glycosidic linkages attached to the polyols, yielding oligosaccharide structures containing from 4 to 10 glucose residues. These new polyols-based oligosaccharides hold promise as innovative prebiotic sweeteners, potentially offering valuable health benefits.

Based on the principle of cascade reaction, a fusion enzyme of dextransucrase and dextranase was designed without linker to catalyze the production of oligo-dextran with homogeneous molecular weight from sucrose in one catalytic step. Due to the different effects of temperature on the two components of the fusion enzyme, temperature served as the "toggle switch" for the catalytic efficiency of the two-level fusion enzyme, regulating the catalytic products of the fusion enzyme. Under optimal conditions, the fusion enzyme efficiently utilized 100 % of the sucrose, and the yield of oligo-dextran with a homogeneous molecular weight reached 70 %. The product has been purified and characterized. The probiotic potential of the product was evaluated by analyzing the growth of 10 probiotic species. Its cytotoxic and anti-inflammatory activities were also determined. The results showed that the long-chain oligo-dextran in this study had significantly better probiotic potential and anti-inflammatory activity compared to other oligosaccharides. This study provides a strategy for the application of oligo-dextran in the food and pharmaceutical industries., Competing Interests: Declaration of competing interest The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Leuconostoc citreum JZ-002 was extracted from artisanal orange wine. This strain was used to synthesize dextran with a purification extraction of 27.9 g/L. The resulting dextran had a molecular weight of 2.45 × 10 6  Da. A significant portion, amounting to 64 % of the structure, is constituted by the main chain, with α-(1,6) glycosidic bonds acting as the linkages. In contrast, the branched chain, comprising 34 % of the entire molecule, is characterized by the presence of α-(1,3) glycosidic bonds. The dextransucrase DsrB, believed to be accountable for the formation of the dextran backbone, was successfully cloned into the pET-28a-AcmA vector. The recombinant expression of the enzyme was achieved. Purified recombinant enzymes and immobilized in a single go using the gram-positive enhancer matrix (GEM). The maximum yield of dextran produced by suchimmobilized enzyme was 191.9 g/L. The composition featured a dextran connected via α-(1,6) glycosidic linkages. Molecular weight controlled synthesis was achieved with sucrose concentrations of 100-2000 mM and enzyme concentrations of 320-1280 U. The Mw of the synthesized dextran extended from 4680 to 1,320,000 Da. By controlling the ratio between enzyme concentration and sucrose concentration, dextrans with diverse Mw can be enzymatically generated., Competing Interests: Declaration of competing interest We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The lignan secoisolariciresinol (SECO) diglucoside (SDG) is a phytoestrogen with diverse effects. LuUGT74S1 glucosylates SECO to SDG, whereby only small amounts of the monoglucoside SMG are formed intermediately, which exhibit increased activity. To identify critical amino acids that are important for enzymatic activity and the SMG/SDG ratio, 3D structural modeling and docking, as well as site-directed mutation studies, were performed. Enzyme assays with ten mutants revealed that four of them had identical kinetic data to LuUGT74S1, while three showed reduced and one increased catalytic efficiency k cat / K m . S82F and E189L substitutions resulted in the complete absence of activity. A17 and Q136 are crucial for the conversion of SMG to SDG as A17S and Q136F mutants exhibited the highest SMG/SDG ratios of 0.7 and 0.4. Kinetic analyses show that diglucosylation is an essentially irreversible reaction, while monoglycosylation is kinetically favored. The results lay the foundation for the biotechnological production of SMG.

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.

Trehalose synthase (TreS) catalyzes the reversible interconversion of maltose to trehalose, playing a vital role in trehalose production. Understanding the catalytic mechanism of TreS is crucial for optimizing the enzyme activity and enhancing its suitability for industrial applications. Here, we report the crystal structures of both the wild type and the E324D mutant of Deinococcus radiodurans trehalose synthase in complex with the trehalose analogue, validoxylamine A. By employing structure-guided mutagenesis, we identified N253, E320, and E324 as crucial residues within the +1 subsite for isomerase activity. Based on these complex structures, we propose the catalytic mechanism underlying the reversible interconversion of maltose to trehalose. These findings significantly advance our comprehension of the reaction mechanism of TreS.

Glycoside phosphorylases are enzymes that are frequently used for polysaccharide synthesis. Some of these enzymes have broad substrate specificity, enabling the synthesis of reducing-end-functionalized glucan chains. Here, we explore the potential of glycoside phosphorylases in synthesizing chromophore-conjugated polysaccharides using commercially available chromophoric model compounds as glycosyl acceptors. Specifically, we report cellulose and β-1,3-glucan synthesis using 2-nitrophenyl β-d-glucopyranoside, 4-nitrophenyl β-d-glucopyranoside, and 2-methoxy-4-(2-nitrovinyl)phenyl β-d-glucopyranoside with Clostridium thermocellum cellodextrin phosphorylase and Thermosipho africanus β-1,3-glucan phosphorylase as catalysts. We demonstrate activity for both enzymes with all assayed chromophoric acceptors and report the crystallization-driven precipitation and detailed structural characterization of the synthesized polysaccharides, i.e., their molar mass distributions and various structural parameters, such as morphology, fibril diameter, lamellar thickness, and crystal form. Our results provide insights for the studies of chromophore-conjugated low molecular weight polysaccharides, glycoside phosphorylases, and the hierarchical assembly of crystalline cellulose and β-1,3-glucan.

Amylosucrase (ASase) efficiently biosynthesizes α-glucoside using flavonoids as acceptor molecules and sucrose as a donor molecule. Here, ASase from Deinococcus wulumuqiensis (DwAS) biosynthesized more naringenin α-glucoside (NαG) with sucrose and naringenin as donor and acceptor molecules, respectively, than other ASases from Deinococcus sp. The biotransformation rate of DwAS to NαG was 21.3% compared to 7.1-16.2% for other ASases. Docking simulations showed that the active site of DwAS was more accessible to naringenin than those of others. The 217th valine in DwAS corresponded to the 221 st isoleucine in Deinococcus geothermalis AS (DgAS), and the isoleucine possibly prevented naringenin from accessing the active site. The DwAS-V217I mutant had a significantly lower biosynthetic rate of NαG than DwAS. The k cat /K m value of DwAS with naringenin as the donor was significantly higher than that of DgAS and DwAS-V217I. In addition, NαG inhibited human intestinal α-glucosidase more efficiently than naringenin., Competing Interests: Declaration of competing interest All of the authors declare no conflicts of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Given the low-calorie, high-sweetness characteristics of steviol glycosides (SGs), developing SGs with improved taste profiles is a key focus. Rebaudioside M8 (Reb M8), a novel non-natural SG derivative obtained through glycosylation at the C-13 position of rebaudioside D (Reb D) using glycosyltransferase UGT94E13, holds promise for further development due to its enhanced sweetness. However, the low catalytic activity of UGT94E13 hampers further research and commercialization. This study aimed to improve the enzymatic activity of UGT94E13 through semirational design, and a variant UGT94E13-F169G/I185G was obtained with the catalytic activity improved by 13.90 times. A cascade reaction involving UGT94E13-F169G/I185G and sucrose synthase At SuSy was established to recycle uridine diphosphate glucose, resulting in an efficient preparation of Reb M8 with a yield of 98%. Moreover, according to the analysis of the distances between the substrate Reb D and enzymes as well as between Reb D and the glucose donor through molecular dynamics simulations, it is found that the positive effect of shortening the distance on glycosylation reaction activity accounts for the improved catalytic activity of UGT94E13-F169G/I185G. Therefore, this study addresses the bottleneck in the efficient production of Reb M8 and provides a foundation for its widespread application in the food industry.

Dextransucrases play a crucial role in the production of dextran from economical sucrose; therefore, there is a pressing demand to explore novel dextransucrases with better performance. This study characterized a dextransucrase enzyme, Lm DexA, which was identified from the Leuconostoc mesenteroides NN710. This bacterium was isolated from the soil of growing dragon fruit in Guangxi province, China. We successfully constructed six different N-terminal truncated variants through sequential analysis. Additionally, a truncated variant, ΔN190 Lm DexA, was constructed by removing the 190 amino acids fragment from the N-terminal. This truncated variant was then successfully expressed heterologously in Escherichia coli and purified. The purified ΔN190 Lm DexA demonstrated optimal hydrolysis activity at a pH of 5.6 and a temperature of 30 °C. Its maximum specific activity was measured to be 126.13 U/mg, with a K m of 13.7 mM. Results demonstrated a significant improvement in the heterologous expression level and total enzyme activity of ΔN190 Lm DexA. ΔN190 Lm DexA exhibited both hydrolytic and transsaccharolytic enzymatic activities. When sucrose was used as the substrate, it primarily produced high-molecular-weight dextran (>400 kDa). However, upon the addition of maltose as a receptor, it resulted in the production of a significant amount of oligosaccharides. Our results can provide valuable information for enhancing the characteristics of recombinant dextransucrase and potentially converting sucrose into high-value-added dextran and oligosaccharides.

Combined cross-linked enzyme aggregates of cyclodextrin glucanotransferase (CGTase) and maltogenic amylase (Mag1) from Bacillus lehensis G1 (Combi-CLEAs-CM) were successfully developed to synthesis maltooligosaccharides (MOS). Yet, the poor cross-linking performance between chitosan (cross-linker) and enzymes resulting low activity recovery and catalytic efficiency. In this study, we proposed the functionalization of cross-linkers with the integration of computational analysis to study the influences of different functional group on cross-linkers in combi-CLEAs development. From in-silico analysis, O-carboxymethyl chitosan (OCMCS) with the highest binding affinity toward both enzymes was chosen and showed alignment with the experimental result, in which OCMCS was synthesized as cross-linker to develop improved activity recovery of Combi-CLEAs-CM-ocmcs (74 %). The thermal stability and deactivation energy (205.86 kJ/mol) of Combi-CLEAs-CM-ocmcs were found to be higher than Combi-CLEAs-CM (192.59 kJ/mol). The introduction of longer side chain of carboxymethyl group led to a more flexible structure of Combi-CLEAs-CM-ocmcs. This alteration significantly reduced the K m value of Combi-CLEAs-CM-ocmcs by about 3.64-fold and resulted in a greater K cat /K m (3.63-fold higher) as compared to Combi-CLEAs-CM. Moreover, Combi-CLEAs-CM-ocmcs improved the reusability with retained >50 % of activity while Combi-CLEAs-CM only 36.18 % after five cycles. Finally, maximum MOS production (777.46 mg/g) was obtained by Combi-CLEAs-CM-ocmcs after optimization using response surface methodology., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Rosli bin Md Illias reports financial support was provided by Universiti Teknologi Malaysia. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Although myricetin (3,3',4',5,5',7-hexahydroxyflavone, MYR) has a high antioxidant capacity and health functions, its use as a functional food material is limited owing to its low stability and water solubility. Amylosucrase (ASase) is capable of biosynthesizing flavonol α-glycoside using flavonols as acceptor molecules and sucrose as a donor molecule. Here, ASase from Deinococcus deserti (DdAS) efficiently biosynthesizes a novel MYR α-triglucoside (MYRαG3) using MYR as the acceptor molecule. Comparative homology analysis and computational simulation revealed that DdAS has a different active pocket for the transglycosylation reaction. DdAS produced MYRαG3 with a conversion efficiency of 67.4 % using 10 mM MYR and 50 mM sucrose as acceptor and donor molecules, respectively. The structure of MYRαG3 was identified as MYR 4'-O-4″,6″-tri-O-α-D-glucopyranoside using NMR and LC-MS. In silico analysis confirmed that DdAS has a distinct active pocket compared to other ASases. In addition, molecular docking simulations predicted the synthetic sequence of MYRαG3. Furthermore, MYRαG3 showed a similar DPPH radical scavenging activity of 49 %, comparable to MYR, but with significantly higher water solubility, which increased from 0.03 μg/mL to 511.5 mg/mL. In conclusion, this study demonstrated the efficient biosynthesis of a novel MYRαG3 using DdAS and highlighted the potential of MYRαG3 as a functional material., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Sucrose phosphorylases, through transglycosylation reactions, are interesting enzymes that can transfer regioselectively glucose from sucrose, the donor substrate, onto acceptors like flavonoids to form glycoconjugates and hence modulate their solubility and bioactivity. Here, we report for the first time the structure of sucrose phosphorylase from the marine bacteria Alteromonas mediterranea (AmSP) and its enzymatic properties. Kinetics of sucrose hydrolysis and transglucosylation capacities on (+)-catechin were investigated. Wild-type enzyme (AmSP-WT) displayed high hydrolytic activity on sucrose and was devoid of transglucosylation activity on (+)-catechin. Two variants, AmSP-Q353F and AmSP-P140D catalysed the regiospecific transglucosylation of (+)-catechin: 89 % of a novel compound (+)-catechin-4'-O-α-d-glucopyranoside (CAT-4') for AmSP-P140D and 92 % of (+)-catechin-3'-O-α-d-glucopyranoside (CAT-3') for AmSP-Q353F. The compound CAT-4' was fully characterized by NMR and mass spectrometry. An explanation for this difference in regiospecificity was provided at atomic level by molecular docking simulations: AmSP-P140D was found to preferentially bind (+)-catechin in a mode that favours glucosylation on its hydroxyl group in position 4' while the binding mode in AmSP-Q353F favoured glucosylation on its hydroxyl group in position 3'., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The amylosucrase (ASase, EC 2.4.1.4) utilizes sucrose as the sole substrate to catalyze multifunctional reactions. It can naturally synthesize α-1,4-linked glucans such as amylose as well as sucrose isomers with more favorable properties than sucrose with a lower intestinal digestibility and non-cariogenic properties. The amino acid sequence of the asase gene from Deinococcus cellulosilyticus (DceAS) exhibits low homology with those of other ASases from other Deinococcus species. In this study, we cloned and expressed DceAS and demonstrated its high activity at pH 6 and pH 8 and maintained stability. It showed higher polymerization activity at pH 6 than at pH 8, but similar isomerization activity and produced more turanose and trehalulose at pH 6 than at pH 8 and produced more isomaltulose at pH 8. Furthermore, the molecular weight of DceAS was 226.6 kDa at pH 6 and 145.5 kDa at pH 8, indicating that it existed as a trimer and dimer, respectively under those conditions. Additionally, circular dichroism spectra showed that the DceAS secondary structure was different at pH 6 and pH 8. These differences in reaction products at different pHs can be harnessed to naturally produce sucrose alternatives that are more beneficial to human health., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper, (Copyright © 2024 Elsevier B.V. All rights reserved.)

The function of polysaccharides is intimately associated with their size, which is largely determined by the processivity of transferases responsible for their synthesis. A tunnel active center architecture has been recognized as a key factor that governs processivity of several glycoside hydrolases (GHs), e.g., cellulases and chitinases. Similar tunnel architecture is also observed in the Limosilactobacillus reuteri 121 GtfB (Lr121 GtfB) α-glucanotransferase from the GH70 family. The molecular element underpinning processivity of these transglucosylases remains underexplored. Here, we report the synthesis of the smallest (α1 → 4)-α-glucan interspersed with linear and branched (α1 → 6) linkages by a novel 4,6-α-glucanotransferase from L. reuteri N1 (LrN1 GtfB) with an open-clefted active center instead of the tunnel structure. Notably, the loop swapping engineering of LrN1 GtfB and Lr121 GtfB based on their crystal structures clarified the impact of the loop-mediated tunnel/cleft structure at the donor subsites -2 to -3 on processivity of these α-glucanotransferases, enabling the tailoring of both product sizes and substrate preferences. This study provides unprecedented insights into the processivity determinants and evolutionary diversification of GH70 α-glucanotransferases and offers a simple route for engineering starch-converting α-glucanotransferases to generate diverse α-glucans for different biotechnological applications.

Despite their broad application potential, the widespread use of β-1,3-glucans has been hampered by the high cost and heterogeneity associated with current production methods. To address this challenge, scalable and economically viable processes are needed for the production of β-1,3-glucans with tailorable molecular mass distributions. Glycoside phosphorylases have shown to be promising catalysts for the bottom-up synthesis of β-1,3-(oligo)glucans since they combine strict regioselectivity with a cheap donor substrate (i.e., α-glucose 1-phosphate). However, the need for an expensive priming substrate (e.g., laminaribiose) and the tendency to produce shorter oligosaccharides still form major bottlenecks. Here, we report the discovery and application of a thermostable β-1,3-oligoglucan phosphorylase originating from Anaerolinea thermophila ( At βOGP). This enzyme combines a superior catalytic efficiency toward glucose as a priming substrate, high thermostability, and the ability to synthesize high molecular mass β-1,3-glucans up to DP 75. Coupling of At βOGP with a thermostable variant of Bifidobacterium adolescentis sucrose phosphorylase enabled the efficient production of tailorable β-1,3-(oligo)glucans from sucrose, with a near-complete conversion of >99 mol %. This cost-efficient process for the conversion of renewable bulk sugar into β-1,3-(oligo)glucans should facilitate the widespread application of these versatile functional fibers across various industries.

The subfamily GH13_16 trehalose synthase (TreS) converts maltose to trehalose and vice versa. Typically, it consists of three domains, but it may contain a C-terminal extension exhibiting clear sequence features of a maltokinase (MaK). The present in silico study was focused on collection of naturally fused TreS-MaKs and their subsequent detailed bioinformatics analysis. Hence a set of total 3354 unique sequences was compared consisting of 1900 single TreSs, 1426 fused TreS-MaKs and 28 single MaKs. Fused TreS-MaKs were divided into five groups, namely with a standard MaK, with mutations in the maltose-binding site, of the catalytic nucleophile, of the general acid/base and of both catalytic residues. Sequence logos bearing the best conserved sequence regions were prepared for both TreSs and MaKs in an effort to find unique sequence features. In addition, linkers connecting the TreS and MaK parts in the fused enzymes were analysed. This analysis revealed that MaKs in fused enzymes have an extended N-terminal regions compared to single MaKs. Finally, the evolutionary relationships were demonstrated by phylogenetic trees of TreS parts from single TreSs and fused TreS-MaKs from the same organism as well as of single TreSs existing in multiple isoforms in the same organism., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Efficient production of cyclodextrins (CDs) has always been challenging. CDs are primarily produced from starch via cyclodextrin glycosyltransferase (CGTase), which acts on α-1,4 glucosidic bonds; however, α-1,6 glucosidic bonds in starch suppress the enzymatic production of CDs. In this study, a glycogen debranching enzyme from Saccharolobus solfataricus STB09 (SsGDE) was utilized to promote the production of β-CD by hydrolyzing α-1,6 glucosidic bonds. The addition of SsGDE (750 U/g of starch) at the liquefaction stage remarkably improved the β-CD yield, with a 43.9% increase. Further mechanism exploration revealed that SsGDE addition could hydrolyze specific branches with less generation of byproducts, thereby promoting CD production. The chain segments of a degree of polymerization ≥13 produced by SsGDE debranching could also be utilized by β-CGTase to convert into CDs. Overall, these findings proposed a new approach of combining SsGDE with β-CGTase to enhance the CD yield.

Of the more than 100 families of glycosyltransferases, family 1 glycosyltransferases catalyze glycosylation using uridine diphosphate (UDP)-sugar as a sugar donor and are thus referred to as UDP-sugar:glycosyl transferases. The blue color of the Nemophila menziesii flower is derived from metalloanthocyanin, which consists of anthocyanin, flavone, and metal ions. Flavone 7-O-β-glucoside-4'-O-β-glucoside in the plant is sequentially biosynthesized from flavons by UDP-glucose:flavone 4'-O-glucosyltransferase (NmF4'GT) and UDP-glucose:flavone 4'-O-glucoside 7-O-glucosyltransferase (NmF4'G7GT). To identify the molecular mechanisms of glucosylation of flavone, the crystal structures of NmF4'G7GT in its apo form and in complex with UDP-glucose or luteolin were determined, and molecular structure prediction using AlphaFold2 was conducted for NmF4'GT. The crystal structures revealed that the size of the ligand-binding pocket and interaction environment for the glucose moiety at the pocket entrance plays a critical role in the substrate preference in NmF4'G7GT. The substrate specificity of NmF4'GT was examined by comparing its model structure with that of NmF4'G7GT. The structure of NmF4'GT may have a smaller acceptor pocket, leading to a substrate preference for non-glucosylated flavones (or flavone aglycones)., Competing Interests: Declaration of competing interest The authors of this publication received partial financial support from Suntory Global Innovation Center, Ltd., according to a collaborative research contract., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Herbicide-resistant weeds are increasingly a problem in crop fields when exposed to similar chemistry over time. To avoid future yield losses, identifying herbicidal chemistry needs to be accelerated. We screened 50,000 small molecules using a liquid-handling robot and light microscopy focusing on pre-emergent herbicides in the family of cellulose biosynthesis inhibitors. Through phenotypic, chemical, genetic, and in silico methods we uncovered 6-{[4-(2-fluorophenyl)-1-piperazinyl]methyl}- N -(2-methoxy-5-methylphenyl)-1,3,5-triazine-2,4-diamine (fluopipamine). Symptomologies support fluopipamine as a putative antagonist of cellulose synthase enzyme 1 (CESA1) from Arabidopsis ( Arabidopsis thaliana ). Ectopic lignification, inhibition of etiolation, phenotypes including loss of anisotropic cellular expansion, swollen roots, and live cell imaging link fluopipamine to cellulose biosynthesis inhibition. Radiolabeled glucose incorporation of cellulose decreased in short-duration experiments when seedlings were incubated in fluopipamine. To elucidate the mechanism, ethylmethanesulfonate mutagenized M2 seedlings were screened for fluopipamine resistance. Two loci of genetic resistance were linked to CESA1. In silico docking of fluopipamine, quinoxyphen, and flupoxam against various CESA1 mutations suggests that an alternative binding site at the interface between CESA proteins is necessary to preserve cellulose polymerization in compound presence. These data uncovered potential fundamental mechanisms of cellulose biosynthesis in plants along with feasible leads for herbicidal uses.