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Many α-agarases have been characterized and are utilized for producing agarooligosaccharides through the degradation of agar and agarose, which are considered valuable for applications in the food and medicine industries. However, the catalytic mechanism and product transformation process of α-agarase remain unclear, limiting further enzyme engineering for industrial applications. In this study, an α-agarase from Catenovulum maritimus STB14 (Cm-AGA) was employed to degrade agarose oligosaccharides (AGOs) with varying degrees of polymerization (DPs) to investigate the catalytic mechanism of α-agarases. The results demonstrated that Cm-AGA could degrade agarose into agarotetraose and agarohexaose. The reducing ends of agarotetraose and agarohexaose spontaneously release unstable 3,6-anhydro-α-l-galactose molecules, which were further degraded into agarotriose and agaropentose. Cm-AGA cannot act on α-1,3-glucoside bonds in agarotriose, agarotetraose, neoagarobiose, and neoagarotetraose but can act on AGOs with a DP greater than four. The product analysis was further verified by β-galactosidase hydrolysis, which specifically cleaves the non-reducing glycosidic bond of agarooligosaccharides. Multiple sequence alignment results showed that two conserved residues, Asp994 and Glu1129, were proposed as catalytic residues and were further identified by site-directed mutagenesis. Molecular docking of Cm-AGA with agaroheptose revealed the potential substrate binding mode of the α-agarase. These findings enhance the understanding of Cm-AGA's catalytic mode and could guide enzyme engineering for modulating the production of agarooligosaccharides. [ABSTRACT FROM AUTHOR]

Debranching enzymes are unable to fully meet the requirements of green production of starch sugars and comprehensive utilization of by-products. Therefore, it is necessary to explore suitable new types of debranching enzymes. This study screened an oligo-1,6-glucosidase gene (oga) from Paenibacillus sp. STB16 and constructed its Bacillus subtilis expression system. This system achieved heterologous expression of the oligo-1,6-glucosidase. The enzymatic properties and substrate specificity of the recombinant oligo-1,6-glucosidase were explored. The results showed that the enzyme activity of the fermentation supernatant of B. subtilis could reach 162.33 U/mL. The recombinant oligo-1,6-glucosidase had the optimum reaction temperature at 50 °C and optimum reaction pH at 6.0. It specifically acted on the &#945-1, 6-glycosidic bonds of branches with degree of polymerization of 1. Compared to other types of debranching enzymes (substrate conversion rate under the detection limit), the recombinant oligo-1,6-glucosidase exhibited stronger hydrolytic activity towards isomaltose, isomaltotriose, and panose. The substrate conversion rate reach 97.4%-100%, with glucose as the product. Therefore, it is more suitable for treating glucose mother liquor. The glucose content in the first mother liquor, second mother liquor and tail liquor of chromatographic separation was significantly increased by 3.6%, 12.7% and 34.4%, respectively. The research results provide a theoretical basis for the development and utilization of new types of debranching enzymes, and also serve as an important reference for the comprehensive utilization of by-products in starch sugar processing. [ABSTRACT FROM AUTHOR]

BACKGROUND Designing a high‐concentration (50%, w/w) maltodextrin saccharification process is a green method to increase the productivity of maltose syrup. RESULTS: In this study, a temperature‐mediated two‐step process using β‐amylase and pullulanase was investigated as a strategy to improve the efficiency of saccharification. During the saccharification process, both pullulanase addition time and temperature adjustment greatly impacted the final maltose yield. These results indicated that an appropriate β‐amylolysis in the first stage (the first 8 h) was required to facilitate saccharification process, with the maltose yield of 8.46% greater than that of the single step saccharification. Molecular structure analysis further demonstrated that a relatively low temperature (50 °C), as compared with a normal temperature (60 °C), in the first stage resulted in a greater number of chains polymerized by at least seven glucose units and a less heterogeneity system within the residual substrate. The molecular structure of the residual substrate might be beneficial for the subsequent cooperation between β‐amylase and pullulanase in the following 40 h (second stage). CONCLUSION: Over a 48 h saccharification, the temperature‐mediated two‐step process dramatically increased the conversion rate of maltodextrin and yielded significantly more maltose and less byproduct, as compared with a constant‐temperature process. The two‐step saccharification process therefore offered an efficient and green strategy for maltose syrup production in industry. © 2020 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Non-digestible oligosaccharides are known to exert health-promoting effects. However, the specific mechanisms by which they regulate host physiology remain unclear. Understanding these mechanisms will facilitate the development of non-digestible oligosaccharide compositions that can achieve synergistic effects. This study selected three representative non-digestible oligosaccharides, namely xylo-oligosaccharides (XOS), galacto-oligosaccharides (GOS), and isomalto-oligosaccharides (IMO), to investigate their effects as dietary interventions on mice fed a high-fat diet. The results demonstrated that XOS and IMO synergistically mitigated weight gain and ectopic lipid deposition. Further analysis revealed that XOS significantly altered the composition of the gut microbiota, while IMO significantly enhanced insulin sensitivity via the PI3K/Akt pathway. Moreover, the combination of XOS and IMO synergistically promoted the oxidation and breakdown of fatty acids and increased the abundance of acetate and propionate-producing bacteria, such as Lactobacillus . These findings suggest a novel strategy for obesity management based on dietary intervention with XOS and IMO.

Molecular structure of starch in daily diet is closely associated with diabetes management. By enzymatically reassembling α-1,4 and α-1,6 glycosidic bonds in starch molecules, we have synthesized an innovative short-clustered maltodextrin (SCMD) which slowly releases glucose during digestion. Here, we investigated the potential benefits of the SCMD-containing diet using diabetic db / db mice. As compared to a diet with normal starch, this dietary style greatly attenuated hyperglycemia and repaired symptoms associated with diabetes. Additionally, in comparison with acarbose (an α-glucosidase inhibitor) administration, the SCMD-containing diet more effectively accelerated brown adipose activation and improved energy metabolism of db / db mice. Furthermore, the SCMD-containing diet was a more suitable approach to improving the intestinal microflora than acarbose administration, especially the proliferation of Mucispirillum , Akkermansia, and Bifidobacterium . These results reveal a novel strategy for diabetes management based on enzymatically rebuilding starch molecules in the daily diet.