The effects of metformin on invertase activity and its inhibition on sucrose digestion were studied. The rapid unfolding kinetics of invertases, followed a two-state model with an inactive intermediate formation. The dynamic interaction between metformin and invertase caused the secondary structure of the enzyme to become less β-sheet, more α-helix, and random coiling oriented, which weakened the binding force between enzyme and its substrate. Metformin acted as a chaotrope and disrupted the hydrogen bonds of water, which facilitated the unfolding of invertase. However, some sugar alcohols, which promoted the H-bond formation of water, could repair the secondary structure of metformin-denatured invertase and therefore regulate the enzyme activity. This research enriches our understanding of the mechanism of enzyme unfolding induced by guanidine compounds. Moreover, because metformin and sugar substitutes are of concern to diabetes, this research also provides useful information for understanding the activity of the digestive enzyme that coexists with metformin and sugar alcohols.

Phenylalanine (Phe) accelerates fruit wound healing by activating phenylpropanoid metabolism. However, whether Phe affects sucrose and respiratory metabolism in fruit during wound healing remains unknown. In this research, we found that preharvest Phe spray promoted sucrose degradation and increased glucose and fructose levels by activating acid invertase (AI), neutral invertase (NI), sucrose synthase (SS) and sucrose phosphate synthase (SPS) on harvested muskmelons. The spray also activated hexokinase (HK), phosphofructokinase (PFK), pyruvate kinase (PK), malate dehydrogenase (MDH), succinate dehydrogenase (SDH) and glucose-6-phosphate dehydrogenase (G6PDH). In addition, the spray improved energy and reducing power levels in the fruit. Taken together, preharvest Phe spray can provide carbon skeleton, energy and reducing power for wound healing by activating the sucrose metabolism, Embden-Meyerhof-Parnas (EMP) pathway, tricarboxylic acid (TCA) cycle and pentose phosphate (PPP) pathway in muskmelon wounds during healing, which is expected to be developed as a new strategy to accelerate fruit wound healing.

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.

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.

In plants, pollen-pistil interactions during pollination and fertilization mediate pollen hydration and germination, pollen tube growth, and seed set and development. Cell wall invertases (CWINs) help provide the carbohydrates for pollen development; however, their roles in pollination and fertilization have not been well established. In cucumber (Cucumis sativus), CsCWIN3 showed the highest expression in flowers, and we further examined CsCWIN3 for functions during pollination to seed set. Both CsCWIN3 transcript and CsCWIN3 protein exhibited similar expression patterns in the sepals, petals, stamen filaments, anther tapetum, and pollen of male flowers, as well as in the stigma, style, transmitting tract, and ovule funiculus of female flowers. Notably, repression of CsCWIN3 in cucumber did not affect the formation of parthenocarpic fruit but resulted in an arrested growth of stigma integuments in female flowers and a partially delayed dehiscence of anthers with decreased pollen viability in male flowers. Consequently, the pollen tube grew poorly in the gynoecia after pollination. In addition, CsCWIN3-RNA interference plants also showed affected seed development. Considering that sugar transporters could function in cucumber fecundity, we highlight the role of CsCWIN3 and a potential close collaboration between CWIN and sugar transporters in these processes. Overall, we used molecular and physiological analyses to determine the CsCWIN3-mediated metabolism during pollen formation, pollen tube growth, and plant fecundity. CsCWIN3 has essential roles from pollination and fertilization to seed set but not parthenocarpic fruit development in cucumber.

Benefiting from our discovery that β-cyclodextrin (β-CD) could enhance the catalytic activity of invertase through hydrogen bonding to improve detection sensitivity, a highly sensitive and convenient biosensor for the detection of miR-21 was proposed, which is based on the simplicity of reading signals from a personal glucose meter (PGM), combined with self-assembled signal amplification probes and the performance of β-CD as an enhancer. In the presence of miR-21, magnetic nanoparticle coupled capture DNA (MNPs-cDNA) could capture it and then connect assist DNA/H1-invertase (aDNA/H1) and self-assembled signal amplification probes (H1/H2) in turn. As a result, a \"super sandwich\" structure was formed. The invertase on MNPs-cDNA could catalyze the hydrolysis of sucrose to glucose and this catalytic process could be enhanced by β-CD. The PGM signal exhibited a linear correlation with miR-21 concentration within the range of 25 pmol L-1 to 3 nmol L-1, and the detection limit was as low as 5 pmol L-1 with high specificity. Moreover, the recoveries were 103.82-124.65% and RSD was 2.59-6.43%. Furthermore, the biosensor was validated for the detection of miR-21 in serum, and the results showed that miR-21 levels in serum samples from patients with Diffuse Large B-Cell Lymphoma (DLBCL) (n = 12) were significantly higher than those from healthy controls (n = 12) (P < 0.001). Therefore, the ingenious combination of PGM-based signal reading, self-assembled signal amplification probes and β-CD as an enhancer successfully constructed a convenient, sensitive and specific biosensing method, which is expected to be applied to clinical diagnosis.

Potato (Solanum tuberosum) is the third most important food crop in the world. Potato tubers must be stored at cold temperatures to minimize sprouting and losses due to disease. However, cold temperatures strongly induce the expression of the potato vacuolar invertase gene (VInv) and cause reducing sugar accumulation. This process, referred to as \"cold-induced sweetening,\" is a major postharvest problem for the potato industry. We discovered that the cold-induced expression of VInv is controlled by a 200 bp enhancer, VInvIn2En, located in its second intron. We identified several DNA motifs in VInvIn2En that bind transcription factors involved in the plant cold stress response. Mutation of these DNA motifs abolished VInvIn2En function as a transcriptional enhancer. We developed VInvIn2En deletion lines in both diploid and tetraploid potato using clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease 9 (Cas9)-mediated gene editing. VInv transcription in cold-stored tubers was significantly reduced in the deletion lines. Interestingly, the VInvIn2En sequence is highly conserved among distantly related Solanum species, including tomato (Solanum lycopersicum) and other non-tuber-bearing species. We conclude that the VInv gene and the VInvIn2En enhancer have adopted distinct roles in the cold stress response in tubers of tuber-bearing Solanum species.

Highly sensitive and facile detection of low levels of protein markers is of great significance for the early diagnosis and efficacy monitoring of diseases. Herein, aided by an efficient tyramine-signal amplification (TSA) mechanism, we wish to report a simple but ultrasensitive immunoassay with signal readout on a portable personal glucose meter (PGM). In this study, the bioconjugates of tyramine and invertase (Tyr-inv), which act as the critical bridge to convert and amplify the protein concentration information into glucose, are prepared following a click chemistry reaction. Then, in the presence of a target protein, the sandwich immunoreaction between the immobilized capture antibody, the target protein, and the horseradish peroxidase (HRP)-conjugated detection antibody is specifically performed in a 96-well microplate. Subsequently, the specifically loaded HRP-conjugated detection antibodies will catalyze the amplified deposition of a large number of Tyr-inv molecules onto adjacent proteins through highly efficient TSA. Then, the deposited invertase, whose dosage can faithfully reflect the original concentration of the target protein, can efficiently convert sucrose to glucose. The amount of finally produced glucose is simply quantified by the PGM, realizing the highly sensitive detection of trace protein markers such as the carcinoembryonic antigen and alpha fetoprotein antigen at the fg/mL level. This method is simple, cost-effective, and ultrasensitive without the requirement of sophisticated instruments or specialized laboratory equipment, which may provide a universal and promising technology for highly sensitive immunoassay for in vitro diagnosis of diseases.