BACKGROUND: The large-scale biocatalytic application of oxidoreductases requires systems for a cost-effective and efficient regeneration of redox cofactors. These represent the major bottleneck for industrial bioproduction and an important cost factor. In this work, co-expression of the genes of invertase and a Baeyer-Villiger monooxygenase from Burkholderia xenovorans to E. coli W ΔcscR and E. coli BL21 (DE3) enabled efficient biotransformation of cyclohexanone to the polymer precursor, ε-caprolactone using sucrose as electron source for regeneration of redox cofactors, at rates comparable to glucose. E. coli W ΔcscR has a native csc regulon enabling sucrose utilization and is deregulated via deletion of the repressor gene (cscR), thus enabling sucrose uptake even at concentrations below 6 mM (2 g L-1). On the other hand, E. coli BL21 (DE3), which is widely used as an expression host does not contain a csc regulon.
RESULTS: Herein, we show a proof of concept where the co-expression of invertase for both E. coli hosts was sufficient for efficient sucrose utilization to sustain cofactor regeneration in the Baeyer-Villiger oxidation of cyclohexanone. Using E. coli W ΔcscR, a specific activity of 37 U gDCW-1 was obtained, demonstrating the suitability of the strain for recombinant gene co-expression and subsequent whole-cell biotransformation. In addition, the same co-expression cassette was transferred and investigated with E. coli BL21 (DE3), which showed a specific activity of 17 U gDCW- 1. Finally, biotransformation using photosynthetically-derived sucrose from Synechocystis S02 with E. coli W ΔcscR expressing BVMO showed complete conversion of cyclohexanone after 3 h, especially with the strain expressing the invertase gene in the periplasm.
CONCLUSIONS: Results show that sucrose can be an alternative electron source to drive whole-cell biotransformations in recombinant E. coli strains opening novel strategies for sustainable chemical production.

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.

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.

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.

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.