Co-immobilization of a bi-enzymatic cascade into hierarchically porous MIL-53 for efficient 6'-sialyllactose production.

6'-Sialyllactose (6'-SL), the most abundant sialylated human milk oligosaccharide, has attracted attention for its potential application in supplementary infant formulas. Herein, we report a facile strategy to construct a cascade bioreactor for the enzymatic synthesis of 6'-SL by co-immobilizing an enzymatic module consisting of CMP-sialic acid synthase and α-2,6-sialyltransferase into hierarchically porous MIL-53 (HP-MIL-53). The as-prepared HP-MIL-53 showed high enzyme immobilization capacity, reaching 226 mg g-1. Furthermore, the co-immobilized enzymes exhibited higher initial catalytic efficiency, and thermal, pH and storage stability than the free ones. Finally, the 6'-SL yield remained >80% after 13 cycles of use. We expect that HP-MIL-53 would have potential industrial applications in the enzymatic modular synthesis of 6'-SL and other glycans.

Extraction, characterization, antioxidation and anti-inflammatory activity of polysaccharides from Bupleurum chinense based on different molecular weights.

Bupleurum chinense polysaccharide has a wide range of biological activities. In this study, Bupleurum chinense polysaccharides (BPs), BPs-1 (30 kDa) and BPs-2 (2000 kDa) with different molecular weights were isolated and prepared by ultrafiltration interception method. The structures of BPs, BPs-1 and BPs-2 were characterised by monosaccharide composition, GC-MS, Fourier transform infra-red spectroscopy and nuclear magnetic resonance. The results showed that the monosaccharide composition of BPs with different molecular weights was the same, but the proportion was different. BPs, BPs-1 and BPs-2 were mainly connected by Glup-(1→,→2,4)-Araf-(1→,→6)-Glup-(1→). The anti-inflammatory activity screening experiment in vitro showed that BPs-1 had stronger anti-inflammatory effect. Antioxidant experiments showed that BPs-2 had high free radical scavenging activity. This study laid a foundation for elucidating the fine structure and structure-activity relationship of Bupleurum chinense polysaccharides and will promote the product development of Bupleurum chinense polysaccharides.

Structural Characterization and Screening for Anti-inflammatory Activity of Polysaccharides with Different Molecular Weights from Astragali Radix.

Astragali Radix polysaccharides (APSs) exhibit a broad spectrum of biological activity, which is mainly related to immune regulation. At present, most available studies focus on total APSs or a certain component of APSs. However, systematic structural study and screening for the anti-inflammatory activity of polysaccharides with different molecular weights (MW) have yet to be conducted. In this study, lipopolysaccharide (LPS)-induced RAW264.7 macrophages were used as a model to investigate the anti-inflammatory activity of APSs and its fractions. The results revealed that fraction APS-I had better anti-inflammatory effects than APS-II. After APS-I was hydrolyzed by trifluoroacetic acid (TFA), the resulting degradation products oligosaccharides were fully methylated. These derivatized oligosaccharides were further analyzed by MALDI-TOF-MS and UPLC-Q-Exactive-MS/MS. The results showed that APS-I was a hetero-polysaccharide with a molecular weight of about 2.0×106 Da, mainly consisting of glucose (46.8 %) and galactose (34.4 %). The degree of polymerization of Astragali Radix oligosaccharides (APOS) was 2-16. APOS were identified as 1,4-glucooligosaccharides and 1,4-galactooligosaccharides. The findings of this study lay the foundation for further elucidation of structure-function relationships of APSs and provide guidance for the development of anti-inflammatory drugs.

Synthesis of acylated derivatives of alginate oligosaccharides and evaluation of their potential antibacterial activities against Staphylococcus aureus.

Alginate oligosaccharides (AOS) are crucial carbohydrate-based biomaterial used in the synthesis of potential drugs and biological agents, but their antibacterial activities are not significant. In this study, AOS acylated derivatives were synthesized by grafting maleic anhydride (MA) onto AOS at varying ratios. Additionally, their inhibitory effects against Staphylococcus aureus were thoroughly investigated. Characterization of the AOS acylated derivatives (AOS-MA-x, where x = 1, 5, 10, and 20) was conducted using Fourier-transformed infrared spectroscopy, 1H nuclear magnetic resonance spectroscopy, and X-ray diffraction, which confirmed the successful synthesis of these derivatives. The bacteriostatic activity of the AOS-MA derivatives was assessed using growth curves and plate coating method, demonstrating significant antibacterial effects against S. aureus, as compared with AOS. Among these derivatives, AOS-MA-20 exhibited the most potent bacteriostatic activity and was selected for further investigation of its inhibitory mechanism. Scanning electron microscopy analysis revealed that treatment with AOS-MA-20 led to the lysis and rupture of S. aureus cells, expelling their intracellular contents. Moreover, AOS-MA-20 disrupted the integrity of cell wall and cell membrane, impacted ATPase activity, and inhibited the formation of biofilm to some extent, ultimately resulting in bacterial death. These findings lay a foundational framework for the development of environmentally friendly antimicrobial agents.

Automated chemoenzymatic modular synthesis of human milk oligosaccharides on a digital microfluidic platform.

Glycans, along with proteins, nucleic acids, and lipids, constitute the four fundamental classes of biomacromolecules found in living organisms. Generally, glycans are attached to proteins or lipids to form glycoconjugates that perform critical roles in various biological processes. Automatic synthesis of glycans is essential for investigation into structure-function relationships of glycans. In this study, we presented a method that integrated magnetic bead-based manipulation and modular chemoenzymatic synthesis of human milk oligosaccharides (HMOs), on a DMF (Digital Microfluidics) platform. On the DMF platform, enzymatic modular reactions were conducted in solution, and purification of products or intermediates was achieved by using DEAE magnetic beads, circumventing the intricate steps required for traditional solid-phase synthesis. With this approach, we have successfully synthesized eleven HMOs with highest yields of up to >90% on the DMF platform. This study would not only lay the foundation for OPME synthesis of glycans on the DMF platform, but also set the stage for developing automated enzymatic glycan synthesizers based on the DMF platform.

Ultra-sensitive fluorescent biosensor for multiple bacteria detection based on CDs/QDs@ZIF-8 and microfluidic fluidized bed.

An ultra-sensitive fluorescent biosensor based on CDs/QDs@ZIF-8 and microfluidic fluidized bed was developed for rapid and ultra-sensitive detection of multiple target bacteria. The zeolitic imidazolate frameworks (ZIF-8) act as the carrier to encapsulate three kinds of fluorescence signal molecules from the CDs/QDs@ZIF-8 signal amplification system. Besides, three kinds of target pathogenic bacteria were automatically, continuously, and circularly captured by the magnetic nanoparticles (MNPs) in the microfluidic fluidized bed. The neutral Na2EDTA solution was the first time reported to not only dissolve the ZIF-8 frameworks from the MNPs-bacteria-CDs/QDs@ZIF-8 sandwich complexes, but also release the CDs/QDs from sandwich complexes with no loss of fluorescence signal. Due to the advantages of signal amplification and automated sample pretreatment, the proposed fluorescent biosensor can simultaneously detect Escherichia coli O157:H7, Salmonella paratyphi A, and Salmonella paratyphi B as low as 101 CFU/mL within 1.5 h, respectively. The mean recovery in spiked milk samples can reach 99.18%, verifying the applicability of this biosensor in detecting multiple bacteria in real samples.

Advances in the preparation, characterization, and biological functions of chitosan oligosaccharide derivatives: A review.

Chitosan oligosaccharide (COS), which represent the positively charged basic amino oligosaccharide in nature, is the deacetylated and degraded products of chitin. COS has become the focus of intensive scientific investigation, with a growing body of practical and clinical studies highlighting its remarkable health-enhancing benefits. These effects encompass a wide range of properties, including antibacterial, antioxidant, anti-inflammatory, and anti-tumor activities. With the rapid advancements in chemical modification technology for oligosaccharides, many COS derivatives have been synthesized and investigated. These newly developed derivatives possess more stable chemical structures, improved biological activities, and find applications across a broader spectrum of fields. Given the recent interest in the chemical modification of COS, this comprehensive review seeks to consolidate knowledge regarding the preparation methods for COS derivatives, alongside discussions on their structural characterization. Additionally, various biological activities of COS derivatives have been discussed in detail. Lastly, the potential applications of COS derivatives in biomedicine have been reviewed and presented.

Unveiling the inverse antimicrobial impact of a hetero-chitooligosaccharide on Candida tropicalis growth and biofilm formation.

Chitosan and chitooligosaccharide (COS) are renowned for their potent antimicrobial prowess, yet the precise antimicrobial efficacy of COS remains elusive due to scant structural information about the utilized saccharides. This study delves into the antimicrobial potential of COS, spotlighting a distinct hetero-chitooligosaccharide dubbed DACOS. In contrast to other COS, DACOS remarkably fosters the growth of Candida tropicalis planktonic cells and fungal biofilms. Employing gradient alcohol precipitation, DACOS was fractionated, unveiling diverse structural characteristics and differential impacts on C. tropicalis. Notably, in a murine model of systemic candidiasis, DACOS, particularly its 70 % alcohol precipitates, manifests a promotive effect on Candida infection. This research unveils a new pathway for exploring the intricate nexus between the structural attributes of chitosan oligosaccharides and their physiological repercussions, underscoring the imperative of crafting chitosan and COS with meticulously defined structural configurations.

Characterization and application of active human α2,6-sialyltransferases ST6GalNAc V and ST6GalNAc VI recombined in Escherichia coli.

Eukaryotic sialyltransferases play key roles in many physiological and pathological events. The expression of active human recombinant sialyltransferases in bacteria is still challenging. In the current study, the genes encoding human N-acetylgalactosaminide α2,6-sialyltransferase V (hST6GalNAc V) and N-acetylgalactosaminide α2,6-sialyltransferase VI (hST6GalNAc VI) lacking the N-terminal transmembrane domains were cloned into the expression vectors, pET-32a and pET-22b, respectively. Soluble and active forms of recombinant hST6GalNAc V and hST6GalNAc VI when coexpressed with the chaperone plasmid pGro7 were successfully achieved in Escherichia coli. Further, lactose (Lac), Lacto-N-triose II (LNT II), lacto-N-tetraose (LNT), and sialyllacto-N-tetraose a (LSTa) were used as acceptor substrates to investigate their activities and substrate specificities. Unexpectedly, both can transfer sialic acid onto all those substrates. Compared with hST6GalNAc V expressed in the mammalian cells, the recombinant two α2,6-sialyltransferases in bacteria displayed flexible substrate specificities and lower enzymatic efficiency. In addition, an important human milk oligosaccharide disialyllacto-N-tetraose (DSLNT) can be synthesized by both human α2,6-sialyltransferases expressed in E. coli using LSTa as an acceptor substrate. To the best of our knowledge, these two active human α2,6-sialyltransferases enzymes were expressed in bacteria for the first time. They showed a high potential to be applied in biotechnology and investigating the molecular mechanisms of biological and pathological interactions related to sialylated glycoconjugates.

In Silico Study on a Binding Mechanism of ssDNA Aptamers Targeting Glycosidic Bond-Containing Small Molecules.

Aptamer-based detection targeting glycoconjugates has attracted significant attention for its remarkable potential in identifying structural changes in saccharides in different stages of various diseases. However, the challenges in screening aptamers for small carbohydrates or glycoconjugates, which contain highly flexible and diverse glycosidic bonds, have hindered their application and commercialization. In this study, we investigated the binding conformations between three glycosidic bond-containing small molecules (GlySMs; glucose, N-acetylneuraminic acid, and neomycin) and their corresponding aptamers in silico, and analyzed factors contributing to their binding affinities. Based on the findings, a novel binding mechanism was proposed, highlighting the central role of the stem structure of the aptamer in binding and recognizing GlySMs and the auxiliary role of the mismatched bases in the adjacent loop. Guided by this binding mechanism, an aptamer with a higher 6'-sialyllactose binding affinity was designed, achieving a KD value of 4.54 ± 0.64 µM in vitro through a single shear and one mutation. The binding mechanism offers crucial guidance for designing high-affinity aptamers, enhancing the virtual screening efficiency for GlySMs. This streamlined workflow filters out ineffective binding sites, accelerating aptamer development and providing novel insights into glycan-nucleic acid interactions.

Ulvan and Ulva oligosaccharides: a systematic review of structure, preparation, biological activities and applications.

Ulva is one of the main green algae causing green tide disasters. Ulvan is the primarily component polysaccharide of the cell wall of Ulva and its complex structure and monosaccharide composition resulted in various biological activities. However, the high-value and effective utilization of extracted ulvan have been obstructed by limitations ranging from large molecular weight and low solubility to poor bioavailability. Ulva oligosaccharide obtained by degrading ulvan can not only ideally retain the various biological activities of ulvan very well but also effectively solve the problems of low solubility and poor bioavailability. The preparation and biological activity studies of ulvan and Ulva oligosaccharides have become a hot spot in the field of marine biological resources development research. At present, the comprehensive reviews of ulvan and Ulva oligosaccharides are still scarce. What are overviewed in this paper are the chemical composition, structure, extraction, and purification of ulvan and Ulva oligosaccharides, where research progress on the biological activities of ulvan and Ulva oligosaccharides is summarized and prospected. A theoretical and practical basis has been provided for further research on ulvan and Ulva oligosaccharides, as well as the high-value development and effective utilization of marine algae resources.