The fecal microbiota of two healthy adults was cultivated in a medium containing commercial fructooligosaccharides [FOS; 1-kestose (GF2), nystose (GF3), and 1F-fructofuranosylnystose (GF4)]. Initially, the proportions of lactobacilli in the two feces samples were only 0.42% and 0.17%; however, they significantly increased to 7.2% and 4.8%, respectively, after cultivation on FOS. Most FOS-utilizing isolates could utilize only GF2; however, Lacticaseibacillus paracasei strain Lp02 could fully consume GF3 and GF4 too. The FOS operon (fosRABCDXE) was present in Lc. paracasei Lp02 and another Lc. paracasei strain, KCTC 3510T, but fosE was only partially present in the non-FOS-degrading strain KCTC 3510T. In addition, the top six upregulated genes in the presence of FOS were fosABCDXE, particularly fosE. FosE is a β-fructosidase that hydrolyzes both sucrose and all three FOS. Finally, a genome-based analysis suggested that fosE is mainly observed in Lc. paracasei, and only 13.5% (61/452) of their reported genomes were confirmed to include it. In conclusion, FosE allows the utilization of FOS, including GF3 and GF4 as well as GF2, by some Lc. paracasei strains, suggesting that this species plays a pivotal role in FOS utilization in the human gut.

Invertases, or β-fructofuranosidases, are metabolic enzymes widely distributed among plants and microorganisms that hydrolyze sucrose and release fructose from various substrates. Invertase was one of the earliest discovered enzymes, first investigated in the mid-nineteenth century, becoming a classical model used in the primary biochemical studies on protein synthesis, activity, and the secretion of glycoproteins. However, it was not until 20 years ago that a member of this family of enzymes was structurally characterized, showing a bimodular arrangement with a β-propeller catalytic domain, and a β-sandwich domain with unknown function. Since then, many studies on related plant and fungal enzymes have revealed them as basically monomeric. By contrast, all yeast enzymes in this family that have been characterized so far have shown sophisticated oligomeric structures mediated by the non-catalytic domain, which is also involved in substrate binding, and how this assembly determines the particular specificity of each enzyme. In this chapter, we will review the available structures of yeast invertases to elucidate the mechanism regulating oligomer formation and compare them with other reported dimeric invertases in which the oligomeric assembly has no apparent functional implications. In addition, recent work on a new family of invertases with absolute specificity for the α-(1,2)-bond of sucrose found in cyanobacteria and plant invertases is highlighted.

The β-fructofuranosidase enzyme from Aspergillus niger has been extensively used to commercially produce fructooligosaccharides from sucrose. In this study, the native and an engineered version of the β-fructofuranosidase enzyme were expressed in Pichia pastoris under control of the glyceraldehyde-3-phosphate dehydrogenase promoter, and production was evaluated in bioreactors using either dissolved oxygen (DO-stat) or constant feed fed-batch feeding strategies. The DO-stat cultivations produced lower biomass concentrations but this resulted in higher volumetric activity for both strains. The native enzyme produced the highest volumetric enzyme activity for both feeding strategies (20.8% and 13.5% higher than that achieved by the engineered enzyme, for DO-stat and constant feed, respectively). However, the constant feed cultivations produced higher biomass concentrations and higher volumetric productivity for both the native as well as engineered enzymes due to shorter process time requirements (59 h for constant feed and 155 h for DO-stat feed). Despite the DO-stat feeding strategy achieving a higher maximum enzyme activity, the constant feed strategy would be preferred for production of the β-fructofuranosidase enzyme using glycerol due to the many industrial advantages related to its enhanced volumetric enzyme productivity.

BACKGROUND: Dendrobium officinale Kimura et Migo, a renowned traditional Chinese orchid herb esteemed for its significant horticultural and medicinal value, thrives in adverse habitats and contends with various abiotic or biotic stresses. Acid invertases (AINV) are widely considered enzymes involved in regulating sucrose metabolism and have been revealed to participate in plant responses to environmental stress. Although members of AINV gene family have been identified and characterized in multiple plant genomes, detailed information regarding this gene family and its expression patterns remains unknown in D. officinale, despite their significance in polysaccharide biosynthesis.
RESULTS: This study systematically analyzed the D. officinale genome and identified four DoAINV genes, which were classified into two subfamilies based on subcellular prediction and phylogenetic analysis. Comparison of gene structures and conserved motifs in DoAINV genes indicated a high-level conservation during their evolution history. The conserved amino acids and domains of DoAINV proteins were identified as pivotal for their functional roles. Additionally, cis-elements associated with responses to abiotic and biotic stress were found to be the most prevalent motif in all DoAINV genes, indicating their responsiveness to stress. Furthermore, bioinformatics analysis of transcriptome data, validated by quantitative real-time reverse transcription PCR (qRT-PCR), revealed distinct organ-specific expression patterns of DoAINV genes across various tissues and in response to abiotic stress. Examination of soluble sugar content and interaction networks provided insights into stress release and sucrose metabolism.
CONCLUSIONS: DoAINV genes are implicated in various activities including growth and development, stress response, and polysaccharide biosynthesis. These findings provide valuable insights into the AINV gene amily of D. officinale and will aid in further elucidating the functions of DoAINV genes.

Cell wall invertase (CIN) is a vital member of plant invertase (INV) and plays a key role in the breakdown of sucrose. This enzyme facilitates the hydrolysis of sucrose into glucose and fructose, which is crucial for various aspects of plant growth and development. However, the function of CIN genes in foxtail millet (Setaria italica) is less studied. In this research, we used the blast-p of NCBI and TBtools for bidirectional comparison, and a total of 13 CIN genes (named SiCINs) were identified from foxtail millet by using Arabidopsis and rice CIN sequences as reference sequences. The phylogenetic tree analysis revealed that the CIN genes can be categorized into three subfamilies: group 1, group 2, and group 3. Furthermore, upon conducting chromosomal localization analysis, it was observed that the 13 SiCINs were distributed unevenly across five chromosomes. Cis-acting elements of SiCIN genes can be classified into three categories: plant growth and development, stress response, and hormone response. The largest number of cis-acting elements were those related to light response (G-box) and the cis-acting elements related to seed-specific regulation (RY-element). qRT-PCR analysis further confirmed that the expression of SiCIN7 and SiCIN8 in the grain was higher than that in any other tissues. The overexpression of SiCIN7 in Arabidopsis improved the grain size and thousand-grain weight, suggesting that SiCIN7 could positively regulate grain development. Our findings will help to further understand the grain-filling mechanism of SiCIN and elucidate the biological mechanism underlying the grain development of SiCIN.

Difructose anhydride I (DFA-I) can be produced from inulin, with DFA-I-forming inulin fructotransferase (IFTase-I). However, the metabolism of inulin through DFA-I remains unclear. To clarify this pathway, several genes of enzymes related to this pathway in the genome of Microbacterium flavum DSM 18909 were synthesized, and the corresponding enzymes were encoded, purified, and investigated in vitro. After inulin is decomposed to DFA-I by IFTase-I, DFA-I is hydrolyzed to inulobiose by DFA-I hydrolase. Inulobiose is then hydrolyzed by β-fructofuranosidase to form fructose. Finally, fructose enters glycolysis through fructokinase. A β-fructofuranosidase (MfFFase1) clears the byproducts (sucrose and fructo-oligosaccharides), which might be partially hydrolyzed by fructan β-(2,1)-fructosidase/1-exohydrolase and another fructofuranosidase (MfFFase2). Exploring the DFA-I pathway of inulin and well-studied enzymes in vitro extends our basic scientific knowledge of the energy-providing way of inulin, thereby paving the way for further investigations in vivo and offering a reference for further nutritional investigation of inulin and DFA-I in the future.

This article examined the effect of geographical (different climate conditions) and floral origins on some quality parameters of honey including the activity of diastase enzyme. Moreover, some non-quality parameters were investigated such as the pH, fructose, glucose, ratio of fructose/glucose and invertase. The honey samples were collected from Asir (cold climate) and Jazan (hot climate) regions at the southwestern part of Saudi Arabia. The geographical origin significantly affected the mean value moisture of the Acacia honey (p-value = 0.02), conductivity of the polyfloral honey (p-value = 0.03), sucrose of the Acacia honey (p-value = 0.02), diastase activity of the Acacia (p-value = 0.001), Ziziphus (p-value = 0.046) and polyfloral honey (p-value ≤ 0.001), fructose of the Acacia honey (p-value = 0.01), glucose of the Ziziphus honey (p-value = 0.03), fructose/ glucose ratio of the Ziziphus honey (p-value = 0.035), and invertase activity of the polyfloral honey (p-value ≤ 0.001). Regarding the effect of the floral origin of the honey from Asir region, the sucrose percentage of the Acacia honey was significantly more than that of the polyfloral honey (p- value = 0.003), the diastase activity of the Acacia honey was significantly more than its activity in the Ziziphus honey (p- value = 0.044), glucose percentage of the Ziziphus honey was significantly more the glucose percentage of the Acacia honey (p-value = 0.009) and the fructose/ glucose ratio of the Ziziphus honey was significantly more than that of the Acacia and polyforal honeys (p-value = 0.011 and p-value = 0.045, respectively). Concerning the significant effects of the floral origin on the quality parameters of the honey samples from Jazan region, the moisture of the Ziziphus honey was significantly increased when compared to the moisture of the Acacia honey (p-value = 0.038), the acidity of the polfloral honey was significantly more than the acidity of the Acacia honey (p-value = 0.049), the sum of fructose and glucose of the polyfloral honey was significantly increased compared to that of the Acacia honey (p-value = 0.015), the pH of the Ziziphus hiney was significantly more than the pH of the polyfloral honey (0.011) and the fructose of the polfloral honey was significantly more than that of the Acacia honey (p-value = 0.031). The effect of the geographical origin of the honey samples on their quality parameters depends on their floral origin and the effect of their floral origin differs according to their geographical origin. This article suggests considering collectively the geographical and floral origins effect when developing honey standards. However, the Codex standards for honey started considering this issue when it changed the standard concentration of HMF in honey from not more than 80-40 mg/Kg for honeys from cold climate and 80 mg/Kg for honeys from hot climates.

Low temperature storage as an alternative to anti-sprouting chemicals in potato storage may induce reducing sugars (RS) accumulation (i.e. glucose and fructose) in potato tubers. This phenomenon is called \"cold induced sweetening\" (CIS) and occurs in certain varieties. CIS leads to a decrease in the organoleptic qualities and darkening of processed potato and the accumulation of toxic molecules such as acrylamide. To identify potato varieties suitable for storage at low temperatures, we screened six commercial processing varieties: Lady Claire (LC), Verdi, Kiebitz (KB), Pirol, Agria and Markies for their CIS characteristics and sprout-forming potential after storage at 4 °C and 8 °C. Our findings reveal that 4 °C storage allows for efficient sprout reduction in all six tested varieties for up to 4.5 months of storage. Three CIS-resistant varieties, namely Verdi, Lady Claire and Kiebitz, were identified as able to be stored for up to four months at 4 °C with limited increase in glucose content. Conversely, Pirol, Agria and Markies showed an increase in glucose content with a decrease in storage temperature and can be considered as CIS-susceptible varieties. After processing into crisps, the CIS-susceptible varieties displayed poor crisp color quality (brown to black color crisps) after storage for two months at 4 °C compared to the storage at 8 °C, whereas the CIS-resistant varieties had good crisp color quality (pale yellow color crisps) after storage at both 4 and 8 °C. Interestingly, the trends of total RS and/or glucose content in the CIS-resistant and in the CIS-susceptible varieties were correlated with the trends in Vacuolar Invertase (VInv) gene expression for most varieties, as well as with the trends in acrylamide content after processing. In addition, reconditioning of Markies variety after storage at 4 °C by gradually increasing the temperature to 15 °C resulted in a significant decrease of VInv transcript levels (reduction of 80 %), acrylamide content (reduction of 75 %) and glucose content when compared to a storage at 4 °C without reconditioning. Those results demonstrate that the reconditioning technique is a key factor for a sustainable potato storage and for improving the quality of processed potatoes.

BACKGROUND: Aspergillus niger ATCC 20611 is an industrially important fructooligosaccharides (FOS) producer since it produces the β-fructofuranosidase with superior transglycosylation activity, which is responsible for the conversion of sucrose to FOS accompanied by the by-product (glucose) generation. This study aims to consume glucose to enhance the content of FOS by heterologously expressing glucose oxidase and peroxidase in engineered A. niger.
RESULTS: Glucose oxidase was successfully expressed and co-localized with β-fructofuranosidase in mycelia. These mycelia were applied to synthesis of FOS, which possessed an increased purity of 60.63% from 52.07%. Furthermore, peroxidase was expressed in A. niger and reached 7.70 U/g, which could remove the potential inhibitor of glucose oxidase to facilitate the FOS synthesis. Finally, the glucose oxidase-expressing strain and the peroxidase-expressing strain were jointly used to synthesize FOS, which content achieved 71.00%.
CONCLUSIONS: This strategy allows for obtaining high-content FOS by the multiple enzymes expressed in the industrial fungus, avoiding additional purification processes used in the production of oligosaccharides. This study not only facilitated the high-purity FOS synthesis, but also demonstrated the potential of A. niger ATCC 20611 as an enzyme-producing cell factory.

Regulation of the luminal pH of late endocytic compartments in continuously fed mammalian cells is poorly understood. Using normal rat kidney fibroblasts, we investigated the reversible assembly/disassembly of the proton pumping V-ATPase when endolysosomes are formed by kissing and fusion of late endosomes with lysosomes and during the subsequent reformation of lysosomes. We took advantage of previous work showing that sucrosomes formed by the uptake of sucrose are swollen endolysosomes from which lysosomes are reformed after uptake of invertase. Using confocal microscopy and subcellular fractionation of NRK cells stably expressing fluorescently tagged proteins, we found net recruitment of the V1 subcomplex during sucrosome formation and loss during lysosome reformation, with a similar time course to RAB7a loss. Addition of invertase did not alter mTORC1 signalling, suggesting that the regulation of reversible V-ATPase assembly/disassembly in continuously fed cells differs from that in cells subject to amino acid depletion/refeeding. Using live cell microscopy, we demonstrated recruitment of a fluorescently tagged V1 subunit during endolysosome formation and a dynamic equilibrium and rapid exchange between the cytosolic and membrane bound pools of this subunit. We conclude that reversible V-ATPase assembly/disassembly plays a key role in regulating endolysosomal/lysosomal pH in continuously fed cells.